Dna methylation based biomarkers for life expectancy and morbidity

ABSTRACT

The disclosure provides seven DNAm-based estimators of plasma protein levels including those of plasminogen activation inhibitor 1 (PAI-1) and growth differentiation factor 15 (GDF15). The predictor of lifespan, DNAm GrimAge (in units of years), is a composite biomarker based on the seven DNAm surrogate markers and a DNAm-based estimator of smoking pack-years. These novel DNAm based biomarkers show the expected relationship with lifestyle factors (including healthy diet or educational attainment) and clinical biomarkers. Overall, these DNAm based biomarkers are expected to find many useful applications including human anti-aging studies.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit under 35 U.S.C. Section 119(e) of co-pending and commonly-assigned U.S. Provisional Patent Application Ser. No. 62/744,010, filed on Oct. 10, 2018, and entitled “DNA METHYLATION BASED BIOMARKERS FOR LIFE EXPECTANCY AND MORBIDITY” which application is incorporated by reference herein.

STATEMENT OF GOVERNMENT INTEREST

This invention was made with government support under Grant Number AG051425, awarded by the National Institutes of Health. The government has certain rights in the invention.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Oct. 3, 2019, is named 30435_0361WOU1_SL.txt and is 161,691 bytes in size.

TECHNICAL FIELD

The invention relates to methods and materials for examining biological aging in individuals.

BACKGROUND OF THE INVENTION

Studies in invertebrates (yeast, worm, flies) have led to a long list of pharmacological agents that promise to intervene in different aspects of the aging process including stress response mimetics, anti-inflammatory interventions, epigenetic modifiers, neuroprotective agents, hormone treatments. While our arsenal of potential anti-aging interventions is brimming with highly promising candidates that delay aging in model organisms, it remains to be seen whether these interventions delay aging in human cells. To facilitate effective in vitro and ex vivo studies, there is a need for robust biomarkers of aging for human fibroblasts and other widely used cell types. One potential biomarker that has gained significant interest in recent years is DNA methylation (DNAm). Chronological time has been shown to elicit predictable hypo- and hyper-methylation changes at many regions across the genome. Several DNAm based biomarkers of aging have been developed including those using blood-based algorithms and the multi-tissue algorithms. These epigenetic age estimators exhibit statistically significant associations with many age-related diseases and conditions.

Recently developed DNA methylation-based biomarkers allow one to estimate the epigenetic age of an individual. For example, the pan tissue epigenetic clock, which is based on 353 dinucleotide markers, known as CpGs (-C-phosphate-G-), can be used to estimate the age of most human cell types, tissues, and organs (Horvath S. DNA methylation age of human tissues and cell types. Genome Biol. 2013: 14(R115). The estimated age, referred to as “DNA methylation age” (DNAm age), correlates with chronological age when methylation is assessed in certain cell types, tissues, and organs including whole blood, brain, breast, kidney, liver, lung, skin and saliva. Other reports described DNAm-based biomarkers that pertain to a single tissue (e.g. saliva or blood). Recent studies suggested that DNAm-based biomarkers of age capture aspects of biological age. For example, we and others have previously shown that individuals whose DNAm age was greater than their chronological age, i.e. individuals who exhibited epigenetic “age acceleration”, were at an increased risk for death from all causes, even after accounting for known risk factors.

There is a need for improved methods of observing phenomena associated with epigenetic aging, independent of chronological age and traditional risk factors of mortality, as well as improved methods for observing the effects of one or more test agents on the epigenetic aging of human cells.

SUMMARY OF THE INVENTION

The invention disclosed herein provides methods and materials designed to observe DNA methylation levels at selected sites within the human genome. Using these methods and materials, embodiments of the invention provide a number of different biomarkers useful for predicting human lifespan, i.e. time to death, based on DNA methylation levels in genomic DNA obtained from samples such as blood tissue, blood cells, saliva, or buccal swabs. As discussed in detail below, embodiments of the invention observe methylation levels at a variety sites within the human genome in order to obtain information on a variety of phenomena associated with aging such as life expectancy, mortality, and morbidity. Disclosure that focuses on the prediction of mortality and morbidity in humans show that these DNAm based biomarkers are highly robust and informative for a range of applications. As discussed in detail below, embodiments of the invention include methods of observing genomic methylation in an individual in order to obtain information on one or more physiological factors associated with an epigenetic age of the individual, as well as methods of observing the effects of one or more test agents on genomic methylation that is associated with the epigenetic aging of human cells.

Embodiments of the invention can be used to provide information that complements and enhances conventional biomarker assessments that are widely used in clinical applications. For example, embodiments of the invention can be used to directly predict/prognosticate mortality, as well as provide further information on a host of age-related conditions such as cardiovascular disease, cancer risk, progression in neurodegeneration, and various measures of frailty. Embodiments of the invention can also be used to estimate the plasma levels of a number of different proteins associated with aging. In addition, one embodiment of the invention can be used to provide highly accurate information on the numbers of cigarettes smoked by an individual in their lifetime.

One biomarker embodiment disclosed herein and referred to as “DNAm GrimAge” is based on DNA methylation measurements at 1113 cytosine-phosphate-guanines (CpG) locations within the human genome. Significantly, this DNAm GrimAge methylation measurement at 1113 locations is collectively based on eight individual DNAm based biomarker assays at selected subsets of CpG locations, subsets of biomarkers that provide information on the numbers of cigarettes smoked by an individual (more precisely smoking packyears) as well as the in vivo plasma levels of: (1) adrenomedullin (ADM); (2) beta-2-microglobulin (B2M); (3) cystatin-C; (4) growth differentiation factor 15 (GDF15); (5) leptin; (6) plasminogen activator inhibitor 1 (PAI1); and (7) tissue inhibitor metalloproteinases 1 (TIMP1). Interestingly, plasma levels of ADM, B2M, cystatin C, and leptin relate to many age-related traits including cognitive functioning. For example, ADM levels are observed to be increased in individuals with hypertension and heart failure. Plasma B2M is a clinical biomarker associated with cardiovascular disease, kidney function, and inflammation. Plasma cystatin-C can be used to assess kidney function. Moreover, GDF-15 is involved in age-related mitochondrial dysfunction and PAI-1 plays a central role in a number of age-related subclinical and clinical conditions (and recent genetic studies link PAI-1 to lifespan).

Embodiments of the invention include, for example, methods of obtaining information on one or more physiological factors associated with an age of an individual. These methods comprise obtaining genomic DNA from the individual, observing methylation of the genomic DNA in at least about 42 methylation markers from the group of methylation markers in SEQ ID NO: 1-SEQ ID NO: 1113, and then correlating observed methylation in the methylation markers with the one or more physiological factors associated with the age of an individual such that information on the one or more physiological factors associated with the age of an individual is obtained. Typically, the genomic DNA is obtained from human fibroblasts, keratinocytes, buccal cells, endothelial cells, lymphoblastoid cells, and/or cells obtained from blood, skin, dermis, epidermis or saliva. In typical embodiments of the invention, methylation is observed by a process comprising treatment of genomic DNA from the population of cells from the individual with bisulfite to transform unmethylated cytosines of CpG dinucleotides in the genomic DNA to uracil; and/or genomic DNA is hybridized to a complimentary polynucleotide sequence disposed on a microarray. Optionally in these embodiments, correlating observed methylation in the methylation markers comprises a regression analysis.

In addition, certain embodiments of the invention observe specific constellations of markers in SEQ ID NO: 1-SEQ ID NO: 1113. For example, in one embodiment of the invention, the one or more physiological factors associated with an age of an individual comprises predicted age or lifespan of an individual, or the time to coronary heart disease in the individual, the method comprising observing methylation of the genomic DNA in about 1113 methylation markers from the group of methylation markers in SEQ ID NO: 1-SEQ ID NO: 1113, and then correlating observed methylation in the about 1113 methylation markers with predicted age or lifespan of an individual, or the time to coronary heart disease in the individual, such that information on predicted age or lifespan of an individual, or the time to coronary heart disease in the individual is obtained. In another embodiment of the invention, the one or more physiological factors associated with an age of an individual comprises a number of years in which the individual has smoked in their lifetime, and the method comprises observing methylation of the genomic DNA in about 172 methylation markers from the group of methylation markers in SEQ ID NO: 1-SEQ ID NO: 172, and then correlating observed methylation in the about 172 methylation markers with the number of years in which an individual has smoked in their lifetime, such that information on the number of years in which the individual has smoked in their lifetime is obtained. In another embodiment of the invention, the one or more physiological factors associated with an age of an individual comprises plasma protein levels of tissue inhibitor metalloproteinase 1 in the individual, and the method comprises observing methylation of the genomic DNA in about 42 methylation markers from the group of methylation markers in SEQ ID NO: 173-SEQ ID NO: 214, and then correlating observed methylation in the about 42 methylation markers with plasma protein levels of tissue inhibitor metalloproteinase 1 in an individual, such that information on plasma protein levels of tissue inhibitor metalloproteinase 1 in an individual is obtained. In another embodiment of the invention, the one or more physiological factors associated with an age of an individual comprises plasma protein levels of Cystatin-C in the individual, the method comprising observing methylation of the genomic DNA in about 87 methylation markers from the group of methylation markers in SEQ ID NO: 215-SEQ ID NO: 301, and then correlating observed methylation in the about 87 methylation markers with plasma protein levels of Cystatin-C in an individual, such that information on plasma protein levels of Cystatin-C in an individual is obtained. In another embodiment of the invention, the one or more physiological factors associated with an age of an individual comprises plasma protein levels of Beta-2-microglobulin in the individual, the method comprising observing methylation of the genomic DNA in about 91 methylation markers from the group of methylation markers in SEQ ID NO: 302-SEQ ID NO: 392, and then correlating observed methylation in the about 91 methylation markers with plasma protein levels of Beta-2-microglobulin in an individual, such that information on plasma protein levels of Beta-2-microglobulin in an individual is obtained. In another embodiment of the invention, the one or more physiological factors associated with an age of an individual comprises plasma protein levels of growth differentiation factor 15 in the individual, the method comprising observing methylation of the genomic DNA in about 137 methylation markers from the group of methylation markers in SEQ ID NO: 393-SEQ ID NO: 529, and then correlating observed methylation in the about 137 methylation markers with plasma protein levels of growth differentiation factor 15 in an individual, such that information on plasma protein levels of growth differentiation factor 15 in an individual is obtained. In another embodiment of the invention, the one or more physiological factors associated with an age of an individual comprises plasma protein levels of adrenomedullin in the individual, the method comprising observing methylation of the genomic DNA in about 186 methylation markers from the group of methylation markers in SEQ ID NO: 530-SEQ ID NO: 715, and then correlating observed methylation in the about 186 methylation markers with plasma protein levels of adrenomedullin in an individual, such that information on plasma protein levels of adrenomedullin in an individual is obtained. In another embodiment of the invention, the one or more physiological factors associated with an age of an individual comprises plasma protein levels of Leptin in the individual, the method comprising observing methylation of the genomic DNA in about 187 methylation markers from the group of methylation markers in SEQ ID NO: 716-SEQ ID NO: 902, and then correlating observed methylation in the about 187 methylation markers with Leptin in an individual, such that information on plasma protein levels of Leptin in an individual is obtained. In another embodiment of the invention, the one or more physiological factors associated with an age of an individual comprises plasma protein levels of plasminogen activator inhibitor 1 in the individual, the method comprising observing methylation of the genomic DNA in about 211 methylation markers from the group of methylation markers in SEQ ID NO: 903-SEQ ID NO: 1113, and then correlating observed methylation in the about 211 methylation markers with plasminogen activator inhibitor 1 in an individual, such that information on plasma protein levels of plasminogen activator inhibitor 1 in an individual is obtained.

Yet another embodiment of the invention is a method of observing the effects of a test agent on genomic methylation associated epigenetic aging of human cells. Typically, the method comprise combining the test agent with human cells, observing methylation status in at least 42 of the methylation markers of SEQ ID NO: 1-SEQ ID NO: 1113 in genomic DNA from the human cells, and then comparing the observations from (b) with observations of the methylation status in at least 42 of methylation markers of SEQ ID NO: 1-SEQ ID NO: 1113 in genomic DNA from control human cells not exposed to the test agent such that effects of the test agent on genomic methylation associated epigenetic aging in the human cells is observed. Typically, the method comprises observing methylation of the genomic DNA in about 172 methylation markers from the group of methylation markers in SEQ ID NO: 1-SEQ ID NO: 172; and/or observing methylation of the genomic DNA in about 42 methylation markers from the group of methylation markers in SEQ ID NO: 173-SEQ ID NO: 214; and/or observing methylation of the genomic DNA in about 87 methylation markers from the group of methylation markers in SEQ ID NO: 215-SEQ ID NO: 301; and/or observing methylation of the genomic DNA in about 91 methylation markers from the group of methylation markers in SEQ ID NO: 302-SEQ ID NO: 392; and/or observing methylation of the genomic DNA in about 137 methylation markers from the group of methylation markers in SEQ ID NO: 393-SEQ ID NO: 529; and/or observing methylation of the genomic DNA in about 186 methylation markers from the group of methylation markers in SEQ ID NO: 530-SEQ ID NO: 715; and/or observing methylation of the genomic DNA in about 187 methylation markers from the group of methylation markers in SEQ ID NO: 716-SEQ ID NO: 902; and/or observing methylation of the genomic DNA in about 211 methylation markers from the group of methylation markers in SEQ ID NO: 903-SEQ ID NO: 1113; and/or observing methylation of the genomic DNA in about 1113 methylation markers of SEQ ID NO: 1-SEQ ID NO: 1113. Optionally in these methods, a plurality of test agents are combined with the human cells in vitro (e.g. primary keratinocytes from a single or, alternatively, multiple donors. In illustrative embodiments of the invention, the test agent is a polypeptide, a polynucleotide or a compound having a molecular weight less than 3,000, 2,000, 1,000 or 500 g/mol.

In certain embodiments of the methods disclosed above, the genomic DNA can be obtained from human fibroblasts, keratinocytes, buccal cells, endothelial cells, lymphoblastoid cells, and/or cells obtained from blood, skin, dermis, epidermis or saliva. In typical embodiments of the invention, methylation is observed by a process comprising treatment of genomic DNA from the population of cells from the individual with bisulfite to transform unmethylated cytosines of CpG dinucleotides in the genomic DNA to uracil. Optionally in such methods, genomic DNA is hybridized to a complimentary sequence disposed on a microarray, and/or genomic DNA is amplified by a polymerase chain reaction process. In some embodiments of the invention, methylation in at least 75% of the respective groups of methylation markers in a constellation of markers is observed (e.g. a constellation of 172 markers, a constellation of 42 markers etc.). In certain embodiments of the invention, correlating observed methylation in the methylation markers comprises a regression analysis, for example one that also considers the sex of the individual, and/or the ethnicity/race of the individual. Optionally for example, correlating observed methylation in the methylation markers comprises a regression analysis selected to predict time to coronary heart disease in the individual.

As discussed below, the DNAm GrimAge biomarker disclosed herein can supplement and enhance existing conventional lifespan predictors. In addition, the subsets of DNAm based biomarkers (e.g. for smoking pack-years and protein plasma levels) have interesting applications in their own right. For example, the DNAm based surrogate biomarker of smoking pack-years could complement self-reported assessments of pack-years. The surprising fact that DNAm pack-years outperforms self-reported pack-years when it comes to lifespan prediction could reflect erroneous self-reporting or true biology: maybe DNAm pack-years is a superior measure of long-term exposure to smoke, or of biological response to it. Moreover, the DNAm based surrogate biomarkers of plasma protein levels disclosed herein can be used, for example, by epidemiologists who have access to stored DNA samples but no access to plasma samples.

Other objects, features and advantages of the present invention will become apparent to those skilled in the art from the following detailed description. It is to be understood, however, that the detailed description and specific examples, while indicating some embodiments of the present invention, are given by way of illustration and not limitation. Many changes and modifications within the scope of the present invention may be made without departing from the spirit thereof, and the invention includes all such modifications.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 provides a schematic of a flowchart for stages of the development and data from the DNAm GrimAge methodology disclosed herein. Surrogate DNAm based biomarkers for smoking pack-years and plasma protein levels were defined and validated using training and test data from the Framingham Heart study (stage 1). Only 12 out of 88 plasma proteins exhibited a correlation r>0.35 with their respective DNAm based surrogate marker in the test data. In stage 2, time-to-death (due to all-cause mortality) was regressed on chronological age, sex, and DNAm based biomarkers of smoking pack-years and the 12 above mentioned plasma protein levels. The ElasticNet regression model automatically selected the following covariates: chronological age (Age), sex (Female), and DNAm based surrogates for smoking pack-years (DNAm PACKYRS), adrenomedullin levels (DNAm ADM), beta-2 microglobulin (DNAm B2M), cystatin C (DNAm Cystatin C), growth differentiation factor 15 (DNAm GDF-15), leptin (DNAm Leptin), plasminogen activation inhibitor 1 (DNAm PAI-1), tissue inhibitor metalloproteinase 1 (DNAm TIMP-1). The linear combination of the covariate values X^(T)β was linearly transformed to be in units of years. Technically speaking DNAm GrimAge is a mortality risk estimator. Metaphorically speaking, it can be interpreted as a DNAm based estimator of biological age or as another epigenetic clock.

FIG. 2 provides a heat map of pairwise correlations of DNAm based biomarkers.

The heat map codes the pairwise Pearson correlations of select variables (surrounding the definition of DNAm GrimAge) in the test data from the Framingham Heart Study (N=625). DNAm GrimAge is defined as a linear combination of chronological age (Age), sex (Female takes on the value 1 for females and 0 otherwise), and eight DNAm based surrogate markers for smoking pack-years (DNAm PACKYRS), adrenomedullin levels (DNAm ADM), beta-2 microglobulin (DNAm B2M), cystatin C (DNAm Cystatin C), growth differentiation factor 15 (DNAm GDF-15), leptin (DNAm Leptin), plasminogen activation inhibitor 1 (DNAm PAI-1), issue inhibitor metalloproteinase 1 (DNAm TIMP-1). The figure also includes an estimator of mortality risk, mortality.res, which can be interpreted as a measure of “excess” mortality risk compared to the baseline risk in the test data. Formally, mortality.res is defined as the deviance residual from a Cox regression model for time-to-death due to all-cause mortality. The rows and columns of the Figure are sorted according to a hierarchical clustering tree. The shades of color (blue, white, and red) visualize correlation values from −1 to 1. Each square reports the Pearson correlation coefficient.

FIGS. 3A-3I show data from meta-analysis forest plots for predicting time-to-death due to all-cause mortality. Each panel reports a meta-analysis forest plot for combining hazard ratios predicting time-to-death based on a DNAm based biomarker (reported in the figure heading) across different strata formed by racial group within cohort. FIG. 3A shows results for AgeAccelGrim. Each row reports a hazard ratio (for time-to-death) and a 95% confidence interval resulting from a Cox regression model in each of 9 strata (defined by cohort and racial groups). Results for (age-adjusted) DNAm based surrogate markers of (FIG. 3B) adrenomedullin (ADM), (FIG. 3C) beta-2 microglobulin (B2M), (FIG. 3D) cystatin C (Cystatin C), (FIG. 3E) growth differentiation factor 15 (GDF-15), (FIG. 3F) leptin, (FIG. 3G) plasminogen activation inhibitor 1 (PAI-1), (FIG. 3H) tissue inhibitor metalloproteinase 1 (TIMP-1) and (FIG. 3I) smoking pack-years (PACKYRS). The sub-title of each panel reports the meta-analysis p-value and a p-value for a test of heterogeneity Cochran Q test (Het.). In FIG. 3A, each hazard ratio (HR) corresponds to a one-year increase in AgeAccelGrim. In FIGS. 3B-3H, each hazard ratio corresponds to an increase in one-standard deviation. In FIG. 3I, hazard ratios correspond to a 1-year increase in pack-years. The most significant meta-analysis P value (here AgeAccelGrim) is marked in red. A non-significant Cochran Q test p-value is desirable because it indicates that the hazard ratios don't differ significantly across the strata. For example, the hazard ratios associated with AgeAccelGrim exhibit insignificant heterogeneity across the strata (Cochran Q test P_(l)s=0.16).

FIGS. 4A-4I show data from meta-analysis forest plots for predicting time-to-coronary heart disease. Each panel reports a meta-analysis forest plot for combining hazard ratios predicting time to CHD and the DNAm based biomarker (reported in the figure heading) across different strata formed by racial groups within cohorts. FIG. 4A shows results for AgeAccelGrim. Each row reports a hazard ratio (for time-to-CHD) and a 95% confidence interval resulting from a Cox regression model in each of 9 strata (defined by cohort and racial groups). Results for (age adjusted) DNAm based surrogate markers of (FIG. 4B) adrenomedullin (ADM), (FIG. 4C) beta-2 microglobulin (B2M), (FIG. 4D) cystatin C (Cystatin C), (FIG. 4E) growth differentiation factor 15 (GDF-15), (FIG. 4F) leptin, (FIG. 4G) plasminogen activation inhibitor 1 (PAI-1), (FIG. 4H) tissue inhibitor metalloproteinase 1 (TIMP-1) and (FIG. 4I) smoking pack-years (PACKYRS). The sub-title of each panel reports the meta-analysis p-value and a p-value for a test of heterogeneity Cochran Q test (Het.). In FIG. 4A, each hazard ratio (HR) corresponds to a one-year increase in AgeAccelGrim. In FIGS. 4B-4H, each hazard ratio corresponds to an increase in one-standard deviation. In FIG. 4I, hazard ratios correspond to a one unit increased in DNAm pack-years. The most significant meta-analysis P value (here AgeAccelGrim) is marked.

FIGS. 5A-5I show data from meta-analysis of associations with total number of age-related conditions. Each panel reports a meta-analysis forest plot for combining regression coefficients between the comorbidity index and the DNAm based biomarker (reported in the figure heading) across different strata, which are formed by racial group within cohort. FIG. 5A shows a meta-analysis of the regression slope between AgeAccelGrim and the comorbidity index. Analogous results for (age adjusted) DNAm based surrogate markers of (FIG. 5B) adrenomedullin (ADM), (FIG. 5C) beta-2 microglobulin (B2M), (FIG. 5D) cystatin C (Cystatin C), (FIG. 5E) growth differentiation factor 15 (GDF-15), (FIG. 5F) leptin, (FIG. 5G) plasminogen activation inhibitor 1 (PAI-1), (FIG. 5H) tissue inhibitor metalloproteinase 1 (TIMP-1) and (FIG. 5I) smoking pack-years (PACKYRS). The individual study results were combined using fixed effect meta-analysis (reported in the panel heading). Cochran Q test for heterogeneity across studies (Het.). The effect sizes correspond to one year of age acceleration in panel A, one pack-year in panel I and one standard deviation in other panels for DNAm proteins. The estimate with the most significant meta P value is marked in red.

FIG. 6 provides a grid showing cross sectional correlations between DNAm biomarkers and lifestyle factors. Robust correlation coefficients (biweight midcorrelation [39]) between 1) AgeAccelGrim and its eight age-adjusted underlying DNAm based surrogate biomarkers and 2) 38 variables including self-reported diet, 9 dietary biomarkers, 12 variables related to metabolic traits and central adiposity, and 5 life style factors. The analysis was performed on the WHI cohort in up to 4200 postmenopausal women.

DETAILED DESCRIPTION OF THE INVENTION

In the description of embodiments, reference may be made to the accompanying figures which form a part hereof, and in which is shown by way of illustration a specific embodiment in which the invention may be practiced. It is to be understood that other embodiments may be utilized, and structural changes may be made without departing from the scope of the present invention. Many of the techniques and procedures described or referenced herein are well understood and commonly employed by those skilled in the art. Unless otherwise defined, all terms of art, notations and other scientific terms or terminology used herein are intended to have the meanings commonly understood by those of skill in the art to which this invention pertains. In some cases, terms with commonly understood meanings are defined herein for clarity and/or for ready reference, and the inclusion of such definitions herein should not necessarily be construed to represent a substantial difference over what is generally understood in the art.

All publications mentioned herein are incorporated herein by reference to disclose and describe aspects, methods and/or materials in connection with the cited publications. For example, Lu et al., Aging (Albany N.Y.). 2019 Jan. 21; 11(2):303-327. doi: 10.18632/aging 101684; PCT Patent Application No.: PCT/US2019/034829, U.S. Patent Publication 20150259742, U.S. patent application Ser. No. 15/025,185, titled “METHOD TO ESTIMATE THE AGE OF TISSUES AND CELL TYPES BASED ON EPIGENETIC MARKERS”, filed by Stefan Horvath; U.S. patent application Ser. No. 14/119,145, titled “METHOD TO ESTIMATE AGE OF INDIVIDUAL BASED ON EPIGENETIC MARKERS IN BIOLOGICAL SAMPLE”, filed by Eric Villain et al.; and Hannum et al. “Genome-Wide Methylation Profiles Reveal Quantitative Views Of Human Aging Rates.” Molecular Cell. 2013; 49(2):359-367 and patent US2015/0259742, are incorporated by reference in their entirety herein.

The invention disclosed herein provides novel and powerful biomarker predictors of life expectancy, mortality, and morbidity based on DNA methylation levels. Our discoveries surrounding the prediction of mortality and morbidity show that the DNAm based biomarkers disclosed herein are highly robust and informative for a range of applications. Embodiments of the DNAm based biomarkers disclosed herein can provide complementary information that enhances and supplements traditional biomarker assessments that are widely used in clinical applications. For example, embodiments of the invention can be used to directly predict/prognosticate mortality, and further information relating to a host of age-related conditions such as cardiovascular disease, cancer risk, progression in neurodegeneration, and various measures of frailty.

Embodiments of the invention include methods of observing the effects of one or more test agents on genomic methylation associated epigenetic aging of human cells. Typically, these methods comprise combining the test agent(s) with human cells (e.g. for specified period of time such as at least 1-7 days, 1-3 weeks, 1-6 months or the like), and then observing methylation status in at least 42 of the methylation markers of SEQ ID NO: 1-SEQ ID NO: 1113 in genomic DNA from the human cells, and then comparing the observations from (b) with observations of the methylation status in at least 42 of methylation markers of SEQ ID NO: 1-SEQ ID NO: 1113 in genomic DNA from control human cells not exposed to the test agent such that effects of the test agent on genomic methylation associated epigenetic aging in the human cells is observed. Typically, the method comprises observing methylation of the genomic DNA in about 172 methylation markers from the group of methylation markers in SEQ ID NO: 1-SEQ ID NO: 172; and/or observing methylation of the genomic DNA in about 42 methylation markers from the group of methylation markers in SEQ ID NO: 173-SEQ ID NO: 214; and/or observing methylation of the genomic DNA in about 87 methylation markers from the group of methylation markers in SEQ ID NO: 215-SEQ ID NO: 301; and/or observing methylation of the genomic DNA in about 91 methylation markers from the group of methylation markers in SEQ ID NO: 302-SEQ ID NO: 392; and/or observing methylation of the genomic DNA in about 137 methylation markers from the group of methylation markers in SEQ ID NO: 393-SEQ ID NO: 529; and/or observing methylation of the genomic DNA in about 186 methylation markers from the group of methylation markers in SEQ ID NO: 530-SEQ ID NO: 715; and/or observing methylation of the genomic DNA in about 187 methylation markers from the group of methylation markers in SEQ ID NO: 716-SEQ ID NO: 902; and/or observing methylation of the genomic DNA in about 211 methylation markers from the group of methylation markers in SEQ ID NO: 903-SEQ ID NO: 1113; and/or observing methylation of the genomic DNA in about 1113 methylation markers of SEQ ID NO: 1-SEQ ID NO: 1113. Optionally in these methods, a plurality of test agents are combined with the human cells in vitro (e.g. primary keratinocytes from a single or, alternatively, multiple donors. In illustrative embodiments of the invention, the test agent is a polypeptide, a polynucleotide or a compound having a molecular weight less than 3,000, 2,000, 1,000 or 500 g/mol.

Embodiments of the invention include a number of different biomarkers useful for predicting human lifespan, i.e. time to death, based on DNA methylation levels in blood tissue, blood cells, saliva, or buccal swabs. One biomarker embodiment, referred to as “DNAm GrimAge”, can be described on two levels. First, it is based on DNA methylation measurements at 1113 locations, called cytosine-phosphate-guanines (CpGs) in the human genome. Second, at a higher level, these DNA methylation measurements at 1113 locations are further based on eight individual DNAm based biomarkers that measure i) the numbers of cigarettes smoked (more precisely smoking packyears) and ii) the plasma levels of the following seven proteins: (1) adrenomedullin (ADM); (2) beta-2-microglobulin (B2M); (3) cystatin-C; (4) growth differentiation factor 15 (GDF15); (5) leptin; (6) plasminogen activator inhibitor 1 (PAI1); and (7) tissue inhibitor metalloproteinases 1 (TIMP1).

Embodiments of the invention further include, for example, methods of obtaining information on one or more physiological factors associated with an age of an individual. These methods comprise obtaining genomic DNA from the individual, observing methylation of the genomic DNA in at least about 42 methylation markers from the group of methylation markers in SEQ ID NO: 1-SEQ ID NO: 1113, and then correlating observed methylation in the methylation markers with the one or more physiological factors associated with the age of an individual such that information on the one or more physiological factors associated with the age of an individual is obtained. Typically, the genomic DNA is obtained from human fibroblasts, keratinocytes, buccal cells, endothelial cells, lymphoblastoid cells, and/or cells obtained from blood, skin, dermis, epidermis or saliva. In typical embodiments of the invention, methylation is observed by a process comprising treatment of genomic DNA from the population of cells from the individual with bisulfite to transform unmethylated cytosines of CpG dinucleotides in the genomic DNA to uracil, and/or genomic DNA is hybridized to a complimentary polynucleotide sequence disposed on a microarray. Optionally in these embodiments, correlating observed methylation in the methylation markers comprises a regression analysis.

In this context, certain embodiments of the invention observe specific constellations of markers in SEQ ID NO: 1-SEQ ID NO: 1113. For example, in one embodiment of the invention, the one or more physiological factors associated with an age of an individual comprises predicted age or lifespan of an individual, or the time to coronary heart disease in the individual, the method comprising observing methylation of the genomic DNA in about 1113 methylation markers from the group of methylation markers in SEQ ID NO: 1-SEQ ID NO: 1113, and then correlating observed methylation in the about 1113 methylation markers with predicted age or lifespan of an individual, or the time to coronary heart disease in the individual, such that information on predicted age or lifespan of an individual, or the time to coronary heart disease in the individual is obtained. In another embodiment of the invention, the one or more physiological factors associated with an age of an individual comprises a number of years in which the individual has smoked in their lifetime, and the method comprises observing methylation of the genomic DNA in about 172 methylation markers from the group of methylation markers in SEQ ID NO: 1-SEQ ID NO: 172, and then correlating observed methylation in the about 172 methylation markers with the number of years in which an individual has smoked in their lifetime, such that information on the number of years in which the individual has smoked in their lifetime is obtained. In another embodiment of the invention, the one or more physiological factors associated with an age of an individual comprises plasma protein levels of tissue inhibitor metalloproteinase 1 in the individual, and the method comprises observing methylation of the genomic DNA in about 42 methylation markers from the group of methylation markers in SEQ ID NO: 173-SEQ ID NO: 214, and then correlating observed methylation in the about 42 methylation markers with plasma protein levels of tissue inhibitor metalloproteinase 1 in an individual, such that information on plasma protein levels of tissue inhibitor metalloproteinase 1 in an individual is obtained. In another embodiment of the invention, the one or more physiological factors associated with an age of an individual comprises plasma protein levels of Cystatin-C in the individual, the method comprising observing methylation of the genomic DNA in about 87 methylation markers from the group of methylation markers in SEQ ID NO: 215-SEQ ID NO: 301, and then correlating observed methylation in the about 87 methylation markers with plasma protein levels of Cystatin-C in an individual, such that information on plasma protein levels of Cystatin-C in an individual is obtained. In another embodiment of the invention, the one or more physiological factors associated with an age of an individual comprises plasma protein levels of Beta-2-microglobulin in the individual, the method comprising observing methylation of the genomic DNA in about 91 methylation markers from the group of methylation markers in SEQ ID NO: 302-SEQ ID NO: 392, and then correlating observed methylation in the about 91 methylation markers with plasma protein levels of Beta-2-microglobulin in an individual, such that information on plasma protein levels of Beta-2-microglobulin in an individual is obtained. In another embodiment of the invention, the one or more physiological factors associated with an age of an individual comprises plasma protein levels of growth differentiation factor 15 in the individual, the method comprising observing methylation of the genomic DNA in about 137 methylation markers from the group of methylation markers in SEQ ID NO: 393-SEQ ID NO: 529, and then correlating observed methylation in the about 137 methylation markers with plasma protein levels of growth differentiation factor 15 in an individual, such that information on plasma protein levels of growth differentiation factor 15 in an individual is obtained. In another embodiment of the invention, the one or more physiological factors associated with an age of an individual comprises plasma protein levels of adrenomedullin in the individual, the method comprising observing methylation of the genomic DNA in about 186 methylation markers from the group of methylation markers in SEQ ID NO: 530-SEQ ID NO: 715, and then correlating observed methylation in the about 186 methylation markers with plasma protein levels of adrenomedullin in an individual, such that information on plasma protein levels of adrenomedullin in an individual is obtained. In another embodiment of the invention, the one or more physiological factors associated with an age of an individual comprises plasma protein levels of Leptin in the individual, the method comprising observing methylation of the genomic DNA in about 187 methylation markers from the group of methylation markers in SEQ ID NO: 716-SEQ ID NO: 902, and then correlating observed methylation in the about 187 methylation markers with Leptin in an individual, such that information on plasma protein levels of Leptin in an individual is obtained. In another embodiment of the invention, the one or more physiological factors associated with an age of an individual comprises plasma protein levels of plasminogen activator inhibitor 1 in the individual, the method comprising observing methylation of the genomic DNA in about 211 methylation markers from the group of methylation markers in SEQ ID NO: 903-SEQ ID NO: 1113, and then correlating observed methylation in the about 211 methylation markers with plasminogen activator inhibitor 1 in an individual, such that information on plasma protein levels of plasminogen activator inhibitor 1 in an individual is obtained. In this context, the DNAm surrogate marker of PAI-1 level stands out when it comes to associations with type 2 diabetes status, glucose-, insulin-, triglyceride levels and measures of adiposity (body mass index and waist-to-hip ratio).

The DNAm GrimAge biomarker disclosed herein outperforms all considered alternative DNAm based predictors of lifespan and time-to-coronary heart disease. The two-stage construction of DNAm GrimAge (via the use of DNAm based surrogate biomarkers for plasma proteins and smoking) is admittedly involved but well worth the effort for the following reason. First, the resulting lifespan predictor outperforms all of the considered alternative biomarkers including those that were constructed by directly regressing lifespan on DNA methylation (such as our novel biomarker DNAm Mortality). Second, DNAm GrimAge outperforms other DNAm based biomarkers when it comes to predicting time to CHD. Third, DNAm GrimAge can be readily interpreted as a linear combination of age, sex, and surrogate biomarkers for plasma proteins and smoking pack-years. Fourth, the DNAm GrimAge estimate has an intuitive interpretation as physiological age since it is in units of years.

We expect that our other surrogate DNAm based biomarkers (for smoking pack-years and PAI-1 levels) will find interesting applications in their own right. We demonstrate that only a small fraction of plasma proteins (we focused on 7 out of 88) can be imputed based on DNA methylation levels. In the Framingham heart study (FHS) data, the measurement of the plasma proteins (exam 7) preceded the measurement of blood methylation data (exam 8) by 6.6 years, which suggests that long term exposure to elevated/decreased plasma levels are associated with subsequent changes in methylation levels. However, the elucidation of cause-and-effect relationships between certain plasma proteins and DNA methylation changes will require future longitudinal cohort studies and mechanistic studies. Our DNAm based surrogate biomarkers of plasma protein levels could also be interesting to epidemiologists who have access to stored DNA samples but no access to plasma samples. Strong literature support links the selected plasma proteins (used in the construction of GrimAge) to various age-related conditions: ADM levels are increased in individuals with hypertension and heart failure. Plasma B2M is a clinical biomarker associated with cardiovascular disease, kidney function, and inflammation. Plasma cystatin-C can be used to assess kidney function. Overall, ADM, B2M, cystatin C, and leptin relate to many age-related traits including cognitive functioning. GDF-15 is involved in age-related mitochondrial dysfunction. PAI-1 plays a central role in a number of age-related subclinical and clinical conditions[9], and recent genetic studies link PAI-1 to lifespan. The tissue inhibitor of metalloproteinases, TIMP-1, plays an anti-apoptotic function.

Despite their obvious strengths, it is unlikely that DNAm based biomarkers will replace existing clinical biomarkers (such as glucose levels, lipid levels, blood pressure) when it comes to informing patient care. Rather, we expected that these biomarkers will complement existing clinical biomarkers when it comes to evaluating anti-aging interventions in vivo and in vitro. Since DNAm captures important properties of the DNA molecule, these DNAm biomarkers are expected to be proximal to innate aging processes.

The DNAm GrimAge biomarker will enhance existing conventional lifespan predictors. Our DNAm based surrogate biomarker of smoking pack-years could complement self-reported assessments of pack-years. The surprising fact that DNAm pack-years outperforms self-reported pack-years when it comes to lifespan prediction could reflect erroneous self-reporting or true biology: maybe DNAm pack-years is a superior measure of long-term exposure to smoke, or of biological response to it.

Beyond lifespan prediction, AgeAccelGrim (and several of its underlying surrogate biomarkers) relate to most age-related conditions (metabolic syndrome, comorbidity, markers of inflammation such as C-reactive protein levels) in the expected way: increased values are associated with a grim condition. Similarly, higher values of AgeAccelGrim (and several DNAm based surrogate markers) are associated with a blood cell composition that is indicative of older individuals and shorter telomere length. While the reported associations are statistically highly significant, the magnitude of the underlying correlations is relatively small. In light of these data, it is unlikely that AgeAccelGrim is simply a marker of immunosenescence. Future studies will need to evaluate the hypothesis that AgeAccelGrim is associated with several hallmarks of aging including immunosenescence, the decline in hematopoietic stem cells, and cell intrinsic epigenetic changes.

While AgeAccelGrim stands out when it comes to lifespan prediction and prediction of time-to-CHD, our (age-adjusted) DNAm surrogate marker of PAI-1 level stands out when it comes to associations with type 2 diabetes status, glucose-, insulin-, triglyceride levels and measures of adiposity (body mass index and waist-to-hip ratio). Inflammation and metabolic conditions are associated with AgeAccelGrim, age-adjusted DNAm PAI-1 and age-adjusted DNAm TIMP-1. Our dietary analysis reveals that vegetable consumption is associated with slower epigenetic aging which echoes previous results. Using the invention disclosed herein, future longitudinal cohort studies can investigate the influence of various stress factors on AgeAccelGrim. Future genetic studies of AgeAccelGrim promise to identify the underlying genetic variants. Overall, our set of novel DNAm based biomarkers has great potential to monitor and evaluate interventions applied to age-related conditions.

Overview of the Two-Stage Approach for Defining DNAm GrimAge

We used a novel two-stage approach for defining a novel DNAm based predictor of life span (time to death due to all cause mortality). In stage 1, we defined a collection of surrogate DNAm biomarkers of physiological risk factors and stress factors. In stage 2, we combined these biomarkers into a single composite biomarker of lifespan (DNAm GrimAge). DNAm GrimAge is calibrated to be in units of years, i.e. it can be interpreted as DNA methylation based estimate of physiological age.

We first sought to develop DNA methylation based surrogate biomarkers of plasma protein levels since plasma proteins such as adrenomedullin, C-reactive protein, plasminogen activation inhibitor 1 (PAI-1), and growth differentiation factor 15 (GDF15) accompany important physiological processes and are associated with age-related conditions [12, 13]. Further, we defined a novel DNAm based estimator of smoking pack-years since smoking is a significant risk factor of mortality and morbidity.

The DNAm based biomarkers defined in stage 1 are attractive surrogate biomarkers for epidemiological studies because a) they lend themselves for imputing plasma protein levels and b) they allow checking self reported questionnaire entries surrounding smoking pack-years based on DNA methylation levels. The surrogate biomarkers have limitations, but it is exciting that stored DNA samples (collected decades ago) will allow one to impute plasma levels for a select group of plasma proteins.

In stage 2, we used these surrogate DNAm biomarkers to define a composite biomarker DNAm GrimAge that outperforms existing DNAm based biomarkers of lifespan. To demonstrate that DNAm GrimAge outperforms existing DNAm based predictors of lifespan we carried out a large-scale meta-analysis (involving more than 7000 Illumina array measurements). We also characterized the resulting DNAm GrimAge estimate with respect to lifestyle factors and a host of age-related conditions, e.g. we demonstrate that our DNAm based biomarkers predict time to cardiovascular disease. Finally, we demonstrate that DNAm GrimAge and several underlying surrogate biomarkers (e.g. DNAm PAI-1, DNAm GDF15) are associated with age-related changes in blood cell composition and leukocyte telomere length.

Practicing the Invention of DNAm GrimAge

To use the epigenetic biomarker, one needs to extract DNA from cells or fluids, e.g. human blood cells, whole blood, peripheral blood mononuclear cells, and saliva (e.g. buccal skin cells). Next, one needs to measure DNA methylation levels in the underlying signature of 1113 CpGs (epigenetic markers) that are being used in the mathematical algorithm. The algorithm leads to an “age” (for each sample or human subject). The higher the value, the higher the risk of death and disease.

FURTHER ILLUSTRATIVE ASPECTS AND EMBODIMENTS OF THE INVENTION

Novel molecular biomarkers of aging, such as those termed “DNAm age”, “epigenetic age” or “apparent methylomic aging rate” allow one to prognosticate mortality, are interesting to gerontologists (aging researchers), epidemiologists, medical professionals, and medical underwriters for life insurances. Exclusively clinical biomarkers such as lipid levels, body mass index, blood pressures have a long and successful history in the life insurance industry. By contrast, molecular biomarkers of aging have rarely been used.

DNA methylation refers to chemical modifications of the DNA molecule. Technological platforms such as the Illumina Infinium microarray or DNA sequencing-based methods have been found to lead to highly robust and reproducible measurements of the DNA methylation levels of a person. There are more than 28 million CpG loci in the human genome. Consequently, certain loci are given unique identifiers such as those found in the Illumina CpG loci database (see, e.g. Technical Note: Epigenetics, CpG Loci Identification ILLUMINA Inc. 2010). These CG locus designation identifiers are used herein. In this context, one embodiment of the invention is a method of obtaining information useful to observe biomarkers associated with a phenotypic age of an individual by observing the methylation status of one or more of the 1113 methylation marker specific GC loci that are identified in Table 9 below.

The term “epigenetic” as used herein means relating to, being, or involving a chemical modification of the DNA molecule. Epigenetic factors include the addition or removal of a methyl group which results in changes of the DNA methylation levels.

The term “nucleic acids” as used herein may include any polymer or oligomer of pyrimidine and purine bases, preferably cytosine, thymine, and uracil, and adenine and guanine, respectively. The present invention contemplates any deoxyribonucleotide, ribonucleotide or peptide nucleic acid component, and any chemical variants thereof, such as methylated, hydroxymethylated or glucosylated forms of these bases, and the like. The polymers or oligomers may be heterogeneous or homogeneous in composition, and may be isolated from naturally-occurring sources or may be artificially or synthetically produced. In addition, the nucleic acids may be DNA or RNA, or a mixture thereof, and may exist permanently or transitionally in single-stranded or double-stranded form, including homoduplex, heteroduplex, and hybrid states.

The term “methylation marker” as used herein refers to a CpG position that is potentially methylated. Methylation typically occurs in a CpG containing nucleic acid. The CpG containing nucleic acid may be present in, e.g., in a CpG island, a CpG doublet, a promoter, an intron, or an exon of gene. For instance, in the genetic regions provided herein the potential methylation sites encompass the promoter/enhancer regions of the indicated genes. Thus, the regions can begin upstream of a gene promoter and extend downstream into the transcribed region.

The term “gene” as used herein refers to a region of genomic DNA associated with a given gene. For example, the region can be defined by a particular gene (such as protein coding sequence exons, intervening introns and associated expression control sequences) and its flanking sequence. It is, however, recognized in the art that methylation in a particular region is generally indicative of the methylation status at proximal genomic sites. Accordingly, determining a methylation status of a gene region can comprise determining a methylation status of a methylation marker within or flanking about 10 bp to 50 bp, about 50 to 100 bp, about 100 bp to 200 bp, about 200 bp to 300 bp, about 300 to 400 bp, about 400 bp to 500 bp, about 500 bp to 600 bp, about 600 to 700 bp, about 700 bp to 800 bp, about 800 to 900 bp, 900 bp to 1 kb, about 1 kb to 2 kb, about 2 kb to 5 kb, or more of a named gene, or CpG position.

The phrase “selectively measuring” as used herein refers to methods wherein only a finite number of methylation marker or genes (comprising methylation markers) are measured rather than assaying essentially all potential methylation marker (or genes) in a genome. For example, in some aspects, “selectively measuring” methylation markers or genes comprising such markers can refer to measuring no more than 211, 187, 137, 91, or 42 different methylation markers or genes comprising methylation markers in Table 9.

The invention described herein provides novel and powerful predictors of life expectancy, mortality, and morbidity based on DNA methylation levels. In this context, it is critical to distinguish clinical from molecular biomarkers of aging. Clinical biomarkers such as lipid levels, blood pressure, blood cell counts have a long and successful history in clinical practice. By contrast, molecular biomarkers of aging are rarely used. However, this is likely to change due to recent breakthroughs in DNA methylation-based biomarkers of aging. Since their inception in 2013, DNA methylation (DNAm) based biomarkers of aging promise to greatly enhance biomedical research, clinical applications, patient care, and even medical underwriting when it comes to life insurance policies and other financial products. They will also be more useful for clinical trials and intervention assessment that target aging, since they are more proximal to the biological changes that characterize the aging process compared to upstream clinical read outs of health and disease status.

It is critical to distinguish clinical from molecular biomarkers of aging. Clinical biomarkers such as lipid levels, blood pressure, blood cell counts have a long and successful history in clinical practice. By contrast, molecular biomarkers of aging are rarely used. However, this is likely to change due to recent breakthroughs in DNA methylation-based biomarkers of aging. Since their inception, DNA methylation (DNAm) based biomarkers of aging promise to greatly enhance biomedical research, clinical applications, patient care, and even medical underwriting when it comes to life insurance policies and other financial products. They will also be more useful for clinical trials and intervention assessment that target aging, since they are more proximal to the biological changes that characterize the aging process compared to upstream clinical read outs of health and disease status.

The disclosure presented herein surrounding the prediction of mortality and morbidity show that DNAm based biomarkers are highly robust and informative for a range of applications. DNAm age can not only be used to directly predict/prognosticate mortality but also relate to a host of age-related conditions such as heart disease risk, cancer risk, dementia status, cardiovascular disease and various measures of frailty. Further embodiments and aspects of the invention are discussed below.

DNA methylation of the methylation markers can be measured using various approaches, which range from commercial array platforms (e.g. from Illumina™) to sequencing approaches of individual genes. This includes standard lab techniques or array platforms. A variety of methods for detecting methylation status or patterns have been described in, for example U.S. Pat. Nos. 6,214,556, 5,786,146, 6,017,704, 6,265,171, 6,200,756, 6,251,594, 5,912,147, 6,331,393, 6,605,432, and 6,300,071 and US Patent Application Publication Nos. 20030148327, 20030148326, 20030143606, 20030082609 and 20050009059, each of which are incorporated herein by reference. Other array-based methods of methylation analysis are disclosed in U.S. patent application Ser. No. 11/058,566. For a review of some methylation detection methods, see, Oakeley, E. J., Pharmacology & Therapeutics 84:389-400 (1999). Available methods include, but are not limited to: reverse-phase HPLC, thin-layer chromatography, SssI methyltransferases with incorporation of labeled methyl groups, the chloracetaldehyde reaction, differentially sensitive restriction enzymes, hydrazine or permanganate treatment (m5C is cleaved by permanganate treatment but not by hydrazine treatment), sodium bisulfite, combined bisulphate-restriction analysis, and methylation sensitive single nucleotide primer extension.

The methylation levels of a subset of the DNA methylation markers disclosed herein are assayed (e.g. using an Illumina™ DNA methylation array or using a PCR protocol involving relevant primers). To quantify the methylation level, one can follow the standard protocol described by Illumina™ to calculate the beta value of methylation, which equals the fraction of methylated cytosines in that location. The invention can also be applied to any other approach for quantifying DNA methylation at locations near the genes as disclosed herein. DNA methylation can be quantified using many currently available assays which include, for example:

a) Molecular break light assay for DNA adenine methyltransferase activity is an assay that is based on the specificity of the restriction enzyme DpnI for fully methylated (adenine methylation) GATC sites in an oligonucleotide labeled with a fluorophore and quencher. The adenine methyltransferase methylates the oligonucleotide making it a substrate for DpnI. Cutting of the oligonucleotide by DpnI gives rise to a fluorescence increase.

b) Methylation-Specific Polymerase Chain Reaction (PCR) is based on a chemical reaction of sodium bisulfite with DNA that converts unmethylated cytosines of CpG dinucleotides to uracil or UpG, followed by traditional PCR. However, methylated cytosines will not be converted in this process, and thus primers are designed to overlap the CpG site of interest, which allows one to determine methylation status as methylated or unmethylated. The beta value can be calculated as the proportion of methylation.

c) Whole genome bisulfite sequencing, also known as BS-Seq, is a genome-wide analysis of DNA methylation. It is based on the sodium bisulfite conversion of genomic DNA, which is then sequencing on a Next-Generation Sequencing (NGS) platform. The sequences obtained are then re-aligned to the reference genome to determine methylation states of CpG dinucleotides based on mismatches resulting from the conversion of unmethylated cytosines into uracil.

d) The Hpall tiny fragment Enrichment by Ligation-mediated PCR (HELP) assay is based on restriction enzymes' differential ability to recognize and cleave methylated and unmethylated CpG DNA sites.

e) Methyl Sensitive Southern Blotting is similar to the HELP assay but uses Southern blotting techniques to probe gene-specific differences in methylation using restriction digests. This technique is used to evaluate local methylation near the binding site for the probe.

f) ChIP-on-chip assay is based on the ability of commercially prepared antibodies to bind to DNA methylation-associated proteins like MeCP2.

g) Restriction landmark genomic scanning is a complicated and now rarely-used assay is based upon restriction enzymes' differential recognition of methylated and unmethylated CpG sites. This assay is similar in concept to the HELP assay.

h) Methylated DNA immunoprecipitation (MeDIP) is analogous to chromatin immunoprecipitation. Immunoprecipitation is used to isolate methylated DNA fragments for input into DNA detection methods such as DNA microarrays (MeDIP-chip) or DNA sequencing (MeDIP-seq).

i) Pyrosequencing of bisulfite treated DNA is a sequencing of an amplicon made by a normal forward primer but a biotinylated reverse primer to PCR the gene of choice. The Pyrosequencer then analyses the sample by denaturing the DNA and adding one nucleotide at a time to the mix according to a sequence given by the user. If there is a mismatch, it is recorded and the percentage of DNA for which the mismatch is present is noted. This gives the user a percentage methylation per CpG island.

In certain embodiments of the invention, the genomic DNA is hybridized to a complimentary sequence (e.g. a synthetic polynucleotide sequence) that is coupled to a matrix (e.g. one disposed within a microarray). Optionally, the genomic DNA is transformed from its natural state via amplification by a polymerase chain reaction process. For example, prior to or concurrent with hybridization to an array, the sample may be amplified by a variety of mechanisms, some of which may employ PCR See, for example, PCR Technology: Principles and Applications for DNA Amplification (Ed. H. A. Erlich, Freeman Press, NY, N.Y., 1992); PCR Protocols: A Guide to Methods and Applications (Eds. Innis, et al., Academic Press, San Diego, Calif., 1990); Mattila et al., Nucleic Acids Res. 19, 4967 (1991); Eckert et al., PCR Methods and Applications 1, 17 (1991); PCR (Eds. McPherson et al., IRL Press, Oxford); and U.S. Pat. Nos. 4,683,202, 4,683,195, 4,800,159, 4,965,188, and 5,333,675. The sample may be amplified on the array. See, for example, U.S. Pat. No. 6,300,070, which is incorporated herein by reference.

In addition to using art accepted modeling techniques on data obtained from embodiments of the invention (e.g. regression analyses), embodiments of the invention can utilize a variety of art accepted technical processes. For example, in certain embodiments of the invention, a bisulfite conversion process is performed so that cytosine residues in the genomic DNA are transformed to uracil, while 5-methylcytosine residues in the genomic DNA are not transformed to uracil. Kits for DNA bisulfite modification are commercially available from, for example, MethylEasy™ (Human Genetic Signatures™) and CpGenome™ Modification Kit (Chemicon™). See also, WO04096825A1, which describes bisulfite modification methods and Olek et al. Nuc. Acids Res. 24:5064-6 (1994), which discloses methods of performing bisulfite treatment and subsequent amplification. Bisulfite treatment allows the methylation status of cytosines to be detected by a variety of methods. For example, any method that may be used to detect a SNP may be used, for examples, see Syvanen, Nature Rev. Gen. 2:930-942 (2001). Methods such as single base extension (SBE) may be used or hybridization of sequence specific probes similar to allele specific hybridization methods. In another aspect the Molecular Inversion Probe (MIP) assay may be used.

The CpG sites discussed herein are found in Table 9 below. The Illumina method takes advantage of sequences flanking a CpG locus to generate a unique CpG locus cluster ID with a similar strategy as NCBI's refSNP IDs (rs#) in dbSNP (see, e.g. Technical Note: Epigenetics, CpG Loci Identification ILLUMINA Inc. 2010).

Example 1: Illustrative Aspects and Working Embodiments of the Invention

DNAm levels have been used to build accurate composite biomarkers of chronological age [1-6]. DNAm-based age (epigenetic age) estimators, include an estimator developed by Horvath [1], based on 353 CpGs in different somatic tissues, and an estimator developed by Hannum [2], based on 71 CpGs in leukocytes. These estimators predict lifespan after adjusting for chronological age and other risk factors [7-9]. Moreover, they are associated with a host of age-related conditions [10-13]. These DNAm-based age estimators highly correlate with chronological age (r>0.9), but they are less accurate in predicting mortality, healthspan, and various age-related morbidities [10]. Several DNAm-based biomarkers are better mortality predictors because they explicitly use lifespan (time to death) in their construction [3, 4]. For example, Zhang et al (2017) combined mortality associated CpGs [4] into an overall mortality risk score. Levine et al (2018) developed a mortality predictor, DNAm PhenoAge, by regressing a phenotypic measure of mortality risk on CpGs [3].

Many analytical strategies are possible for building mortality predictors from DNAm data. The direct approach involves regressing time-to-death (due to all-cause mortality) on DNAm levels. Here we describe a two-stage procedure: In stage 1, we defined DNAm-based surrogate biomarkers of smoking pack-years and select plasma proteins which have been associated with mortality or morbidity. In stage 2, we regressed time-to-death on these DNAm based surrogate biomarkers. The mortality risk estimate of the regression model is linearly transformed so that it is in units of age. We coin this DNAm-based biomarker of mortality “DNAm GrimAge” because high values are grim news when it comes to lifespan. Using validation data from three ethnic groups, we demonstrate that DNAm GrimAge stands out in terms of its predictive ability for time-to-death due to all-cause mortality and time-to-coronary heart disease. An age-adjusted version of DNAm GrimAge, which can be regarded as a new measure of epigenetic age acceleration (AgeAccelGrim), is associated with a host of age-related conditions, lifestyle factors, biomarkers of immunosenescence, and leukocyte telomere length.

Overview of the Two-Stage Approach for Defining DNAm GrimAge

We constructed the DNAm GrimAge in two-stages. First, we defined surrogate DNAm biomarkers of physiological risk factors and stress factors. These include the following plasma proteins: adrenomedullin, C-reactive protein, plasminogen activation inhibitor 1 (PAI-1), and growth differentiation factor 15 (GDF15) [14, 15]. In addition, given that smoking is a significant risk factor of mortality and morbidity, we also used DNAm-based estimator of smoking pack-years. Second, we combined these biomarkers into a single composite biomarker of lifespan, DNAm GrimAge, which is expressed in units of years. We then performed a large-scale meta-analysis (involving more than 7000 Illumina array measurements), showing that DNAm GrimAge is a better predictor of lifespan that present DNAm-based predictors.

Our studies reveal a surprising finding: sometimes, the DNAm based surrogate biomarkers (e.g. for smoking pack-years) is a better predictors of mortality than the underlying observed (self-reported) biomarker. We also correlated DNAm GrimAge with lifestyle factors and a host of age-related conditions, e.g. we demonstrate that our DNAm based biomarkers predict time to cardiovascular disease. Finally, we demonstrate that DNAm GrimAge is associated with age-related changes in blood cell composition and leukocyte telomere length.

Training and Test Data from the Framingham Heart Study

We correlated levels of 88 plasma protein variables (measured using an immunoassay) with DNAm array data generated from the same blood samples of n=2,356 individuals from the Framingham heart study (FHS) Offspring Cohort [16] (note 1 below). We randomly split the FHS data into a training set (70% of the FHS pedigrees, N=1731 individuals from 622 pedigrees) and a test data set (30% pedigrees, N=625 individuals from 266 pedigrees, (Table 3). The mean age of individuals donating DNA for the training set was 66 years. The mean age of individuals in the test dataset was 67. These participants had a similar demographic profile, smoking history, and number of years' follow-up as those used for the training set (Table 3).

Stage 1: DNAm-Based Surrogate Biomarkers of Plasma Proteins and Smoking Pack-Years

We used the training data to define DNAm-based surrogate markers of 88 plasma protein variables and smoking pack-years. We restricted the analysis to CpGs that are present on both the Illumina Infinium 450K array and the new Illumina EPIC methylation array in order to ensure future compatibility. Each of the 88 plasma protein variables (dependent variable) was regressed on chronological age, sex, and the CpGs levels in the training data using an elastic net regression model, which automatically selected a subset of CpGs (typically fewer than about 200 CpGs) whose linear combination best predicted the corresponding plasma level in the training data (Methods). For example, the DNAm levels of 137 CpGs and 211 CpGs allowed us to estimate the plasma levels of GDF15 and PAI-1, respectively. The predicted DNAm values of GDF15 and PAI-1 can be used as surrogate markers for the observed plasma levels. In general, we denote DNAm-based surrogate markers of plasma proteins and smoking pack-years by adding the prefix “DNAm” to the respective variable name, e.g. DNAm pack-years (FIG. 1 and Table 4).

Not all of the available 88 plasma protein could be successfully imputed based on DNAm data. Only 12 (out of 88) plasma proteins exhibited a correlation coefficient greater than 0.35 between the observed plasma levels and their respective DNAm-based surrogate marker in the test data set, (Table 1). We focused on these 12 DNAm surrogate biomarkers in stage 2. Additionally, we constructed a DNAm-based surrogate of self-reported smoking pack-years, DNAm pack-years, based on a linear combination of 172 CpGs.

Stage 2: Constructing a Composite Biomarker of Lifespan Based on Surrogate Biomarkers

In stage 2, we developed a predictor of mortality by regressing time-to-death due to all-cause mortality (dependent variable) on the following covariates: the DNAm-based estimator of smoking pack-years, chronological age at the time of the blood draw, sex, and the 12 DNAm-based surrogate biomarkers of plasma protein levels. The ElasticNet Cox regression model automatically selected the following covariates: DNAm pack-years, age, sex, and the following 7 DNAm based surrogate markers of plasma proteins: adrenomedullin (ADM), beta-2-microglobulim (B2M), cystatin C (Cystatin C), GDF-15, leptin (Leptin), PAI-1, and tissue inhibitor metalloproteinases 1 (TIMP-1), (Table 4). DNAm-based biomarkers for smoking pack-years and the 7 plasma proteins are based on typically fewer than about 200 CpGs each, totaling 1,113 unique CpGs (Table 4). Details on the plasma proteins can be found in Note 2 below.

The linear combination of covariates resulting from the ElasticNet Cox regression model can be interpreted as an estimate of the logarithm of the hazard ratio of mortality. We linearly transformed this parameter into an age estimate, i.e., DNAm GrimAge, by performing a linear transformation whose slope and intercept terms were chosen by forcing the mean and variance of DNAm GrimAge to match that of chronological age in the training data (Methods, FIG. 1). In independent test data, DNAm GrimAge is calculated without estimating any parameter because the numeric values of all parameters were chosen in the training data. Following the terminology from previous articles on DNAm-based biomarkers of aging, we defined a novel measure of epigenetic age acceleration, AgeAccelGrim, which, by definition, is not correlated (r=0) with chronological age. Toward this end, we regressed DNAm GrimAge on chronological age using a linear regression model and defined AgeAccelGrim as the corresponding raw residual (i.e. the difference between the observed value of DNAm age minus its expected value). Thus, a positive (or negative) value of AgeAccelGrim indicates that the DNAm GrimAge is higher (or lower) than expected based on chronological age.

Unless indicated otherwise, we used AgeAccelGrim (rather than DNAm GrimAge) in association tests of age-related conditions because age was a confounder in these analyses. For the same reason, we also used age-adjusted versions of our DNA based surrogate markers (for smoking pack-years and the seven plasma protein levels). In general, all association tests were adjusted for chronological age and, when required, other confounders (such as sex, Methods).

Pairwise Correlations Between DNAm GrimAge and Surrogate Biomarkers

Using the test data from the FHS, we calculated pairwise correlations between DNAm GrimAge and its underlying variables (FIG. 2 and Table 4). DNAm GrimAge is highly correlated with DNAm TIMP-1 (r=0.90) and chronological age (r=0.82). An estimate of excess mortality risk (called mortality residual mortality.res) exhibits higher positive correlations with both DNAm GrimAge and DNAm TIMP_1 (r˜0.40) than with chronological age (r˜0.35, FIG. 2), in keeping with our later finding that these DNAm biomarkers are better predictors of lifespan than chronological age. With the exception of DNAm Leptin, all of the DNAm based biomarkers exhibited positive correlations with the measure of excess mortality risk (0.41≥r≥0.16, FIG. 2). With the exception of DNAm Leptin, all DNAm based surrogate biomarkers exhibited moderate to strong pairwise correlations with each other.

DNAm Leptin is elevated in females consistent with what has been reported in the literature [17, 18]. After stratifying by sex, we find that plasma leptin levels increase weakly with age (r=0.18 and P=2.1E-3 in males; r=0.19, P=4.8E-4 in females).

Predicting Time-to-Death in Validation Data

To evaluate whether our novel DNAm based biomarkers are better predictors of lifespan than chronological age, we analyzed N=7,375 Illumina methylation arrays generated from blood samples of 6,935 individuals comprising 3 ethnic/racial groups: 50% European ancestry (Caucasians), 40% African Americans, and 10% Hispanic ancestry (Table 2, Methods). The data came from different cohort studies: test data from the FHS, BA23 and EMPC study from the Women's Health Initiative (WHI), the InCHIANTI cohort study, and African Americans from the Jackson Heart Study (JHS). We stratified each cohort by race/ethnicity (resulting in 9 strata) to avoid confounding and to ascertain whether the mortality predictors apply to each group separately.

The mean chronological age at the time of the blood draw was 63.0 years. The mean follow-up time (used for assessing time-to-death due to all-cause mortality) was 13.7 years. Since chronological age is one of the component variables underlying DNAmGrimAge, it is not surprising that the latter is highly correlated with age in each of the study cohorts (r≥0.79).

While each (age-adjusted) component variable underlying DNAm GrimAge is a significant predictor of lifespan (FIG. 3), DNAm pack-years (meta-analysis P=1.7E-47) and DNAm PAI-1(P=5.4E-28) exhibit the most significant meta-analysis P-values. The fixed effects meta-analysis P-values reveal that AgeAccelGrim stands out when it comes to lifespan prediction (meta-analysis P=2.0E-75, FIG. 3A). The same applies when the analysis is restricted to never-smokers or to former/current smokers. AgeAccelGrim remains a highly significant predictor of lifespan after restricting the analysis to never-smokers (N=3,988, meta-analysis P=1.1E-16) or to former/current smokers (P=3.5E-33).

DNAm Based Surrogates Sometimes Outperform Observed Biomarkers

Our DNAm-based surrogate biomarker for smoking pack-years has to surprising properties. First, it predicts lifespan in never-smokers (P=1.6E-6). Second, the surrogate marker is a more significant predictor of lifespan than self-reported pack-years: P=8.5E-5 for DNAm marker versus P=2.1E-3 for observed pack-years in in the FHS test data; similarly, P=5.3E-4 versus 0.18 in the InChianti Study (Table 5). The superior predictive performance of DNAm based surrogate biomarkers vis-à-vis their observed counter parts also applies to PAI-1 plasma levels (P=8.7E-4 for the DNAm marker versus P=0.074 for the observed levels), TIMP-1 (P=3.8E-4 for the DNAm marker versus P=0.017), and to a lesser extent to cystatin C (P=0.019 for the DNAm estimator versus P=0.054 for the observed level, (Table 6).

Mortality Prediction Based on Observed Plasma Protein Levels

The AgeAccelGrim is a composite biomarker derived from DNAm-based surrogate biomarkers of plasma protein levels and smoking pack-years. This begs the question whether a predictor of lifespan based on observed plasma protein levels and self-reported smoking pack-years, outperforms its DNAm-based analog. Analogous to our construction of DNAm GrimAge, we used a Cox regression model to regress-time to-death on the observed plasma protein levels and self-reported pack-year in the training data (Methods). The resulting mortality risk estimator (defined as weighted average of the observed biomarkers) was linearly transformed into units of years. The resulting predictor, i.e., observed GrimAge, and its age-adjusted version. i.e., observed AgeAccelGrim, were compared in the FHS, showing similar HRs (observed AgeAccelGrim HR=1.10, P=3.2E-7; observed AgeAccelGrim HR=1.12, P=8.6E-5, Table 7). Overall, this comparison shows that DNAm levels in general and our DNAm-based surrogate biomarkers in particular capture a substantial proportion of the information captured by the 7 selected plasma proteins and self-reported smoking pack-years. Since our study focuses on DNAm based biomarkers, we will only consider DNAm based biomarkers in the following.

Age-Related Conditions

Our Cox regression analysis of time to coronary heart disease (CHD), reveals that AgeAccelGrim is highly predictive of incident CHD (HR=1.07, P=6.2E-24 and P_(I)z=0.4, FIG. 4A). As expected, several underlying DNAm-based surrogate biomarkers also predict incident CHD notably our age-adjusted versions of DNAm smoking pack-years (HR=1.02, P=6.4E-14) and DNAm PAI-1 (HR=1.31 per SD, P=3.6E-12). Similarly, time-to-congestive heart failure (CHF) is associated with AgeAccelGrim (HR=1.10 and P=4.9E-9), age adjusted DNAm cystatin C (HR=2.02 and P=2.0E-10) and DNAm PAI-1 (HR=1.58 and P=8.9E-10).

Cross sectional studies reveal that AgeAccelGrim is associated with hypertension (odds ratio [OR]=1.04 and P=5.1E-13), type 2 diabetes (OR=1.02 and P=0.01), and physical functioning (Stouffer P=3.8E-13). All of the reported associations are in the expected directions, e.g. higher values of AgeAccelGrim are associated with lower physical functioning levels. In women, early age at menopause is associated with significantly higher values of AgeAccelGrim (P=1.6E-12) and to a lesser extent with all of the age-adjusted versions of the DNAm based surrogate markers, notably DNA cystatin C (P=2.2E-6) and DNAm GDF-15 (P=1.3E-5).

DNAm Plasminogen Activation Inhibitor I

AgeAccelGrimAge outperforms (age-adjusted versions of) DNAm smoking pack-years and the 7 DNAm-based surrogate markers of plasma protein levels when it comes to predicting time-to-death or time-to-coronary heart disease (FIGS. 3 & 4). However, age-adjusted DNAm PAI-1 outperforms AgeAccelGrim for several age-related traits, notably the comorbidity index (defined as total number of age-related conditions) where Stouffer's meta-analysis P value for DNAm PAI-1 (P=1.1E-46) is more significant than that for AgeAccelGrim (P=1.2E-15, FIG. 5). Similar to AgeAccelGrim, higher levels of age-adjusted DNAm PAI-1 are associated with hypertension status, type 2 diabetes status, time-to-CHD (FIG. 4), time-to-CHF, and early age at menopause and lower levels are associated with disease free status (Stouffer P=2.9E-11) and better physical functioning (Stouffer P=1.2E-12).

Heritability Analysis

We used pedigree based polygenic models (Methods) to arrive at heritability estimates of AgeAccelGrim and our other biomarkers. Significant heritability estimates can be observed for AgeAccelGrim (h²=0.30, P=0.022) and observed AgeAccelGrim (h²=0.37, P=0.0060, Table 8). Similarly, several of our DNAm based surrogate biomarkers (for PAI1, B2M, ADM, GDF15) and their observed counterparts are highly heritable (Table 8), e.g. DNAm PAI-1 (h²=0.34 and P=7.1E-3), observed PAI-1 levels (h²=0.51, P=6.2E-4), DNAm Beta 2 microglobulin levels (h²=0.45, P=2.4E-3), and observed B2M (h²=0.34, P=3.3E-3). Overall, these results show that many DNAm based biomarkers are under considerable genetic control.

AgeAccelGrim Versus Other Epigenetic Measures of Age Acceleration

Using the same validation datasets (N=7,375 arrays), we compared DNAm GrimAge with three widely-used DNA-based biomarkers of aging: DNAm age estimator based on different somatic tissues by Horvath (2013) [1],

The DNAm age estimator based on leukocytes by Hannum (2013) [2] and the DNAm PhenoAge estimator by Levine (2018) [3]. The respective age adjusted measures of epigenetic age acceleration will be denoted as AgeAccel (or AgeAccelerationResidual), AgeAccelHannum, and AgeAccelPheno following the notation of previous publications. The four epigenetic measures of age acceleration (including AgeAccelGrim) are in units of year. AgeAccelGrim exhibits moderate positive correlations with each of the three alternative measures of epigenetic age acceleration (0.17≤r≤0.45) and the strongest correlation with AgeAccelPheno. AgeAccelGrim stands out with respect to the meta-analysis P-values for predicting time-to-death: AgeAccelGrim (P=2.0E-75, HR=1.10), AgeAccel (Meta P=8.9E-5, HR=1.02), AgeAccelHannum (Meta P=6.8E-16, HR=1.04), AgeAccelPheno (Meta P=3.5E-36, HR=1.05). The results remain qualitatively the same after restricting the analysis to never-smokers or former/current smokers.

Similarly, AgeAccelGrim stands out when comparing individuals in the top 20% percentile of epigenetic age acceleration to those in the bottom 20% percentile (Stouffer meta-analysis P=2.7E-39), AgeAccelPheno (P=3.0E-20), AgeAccelHannum (P=4.9E-6), and AgeAccel (P=0.14).

When it comes to significant associations with the comorbidity index, age-adjusted DNAm PAI-1(P_(DNAmPAI-1)=1.1E-46, FIG. 5) outperforms all other DNAm-based biomarkers including AgeAccelGrim (P_(AgeAccelGrim)=1.2E-15) and AgeAccelPheno (P_(AgeAccelPheno)==2.7E-18).

AgeAccelGrim is more informative than AgeAccelPheno when it comes to predicting time to CHD (P_(AgeAccelOrtm)=6.2E-24 and HR_(AgeAccelGrim)=1.07 versus P_(AgeAccelPheno)=1.7E-8 and HR_(AgeAccelPheno)=1.03) even after stratifying the analysis by smoking status.

AgeAccelGrim greatly outperforms the other 3 measures of epigenetic age acceleration including predicting time to (any) cancer (AgeAccelGrim P=1.3E-12 versus AgeAccelPheno P=2.7E-3) and as related to an inverse association with early age at menopause in women (AgeAccelGrim P=1.6E-12 versus AgeAccel P=2.2E-3). A sensitivity analysis reveals that the latter finding remains qualitatively the same even after removing the InChianti cohort, which exhibited the strongest negative association between epigenetic age acceleration and age at menopause.

Multivariate Cox Models Adjusting for Traditional Risk Factors

Our above-mentioned Cox regression models adjusted for age at blood draw (baseline), batch, pedigree, and intra subject correlation as needed. We also fit multivariate Cox regression models that included additional covariates assessed at baseline: body mass index, educational level, alcohol intake, smoking pack-years, prior history of diabetes, prior history of cancer, and hypertension status (Methods). Even after adjusting for these known risk factors for morbidity, AgeAccelGrim remained a highly significant predictor of lifespan (P=5.7E-29) and time-to-CHD (P=3.7E-11) and outperformed previously published measures of epigenetic age acceleration.

Stratified Analyses

We evaluated AgeAccelGrim and underlying DNAm biomarkers in different strata characterized by age (younger/older than 65 years), body mass index (obese versus non-obese), educational attainment, prevalent condition at baseline such as prior history of cancer, type 2 diabetes, or hypertension. In all of these strata, AgeAccelGrim remains a significant predictor of time-to-death and time-to-CHD. Further, AgeAccelGrim outperforms existing DNAm based biomarkers of aging in all strata except for one (comprised of n=281 individuals with a prior history of cancer).

These subgroup analysis results also confirm that epigenetic age acceleration is an independent predictor of earlier mortality even after adjusting for possible confounders and within major subgroups of the population. With few exceptions, we found that DNAm based PA-1, TIMP-1 and pack-years remained highly significant in each stratum.

Exceptionally Fast/Slow Agers

The DNAm GrimAge estimate has an intuitive interpretation as physiological age since it is in units of years. However, if someone is 8 years older than expected, that does not mean that this person has on average an 8-year shorter life expectancy. Rather, lifespan calculations need to account for the hazard ratio associated with AgeAccelGim as indicated in the following. It is a statistical co-incidence that the hazard ratio associated with one-year increase in AgeAccelGrim is the same in strata comprised of never-smokers (HR=1.10), former/current smokers (HR=1.10), and among all individuals combined (HR=1.10). This allows us to evaluate the mortality risks in exceptionally fast and slow agers (according to AgeAccelGrim) irrespective of their smoking status. The top 5^(th) percentile and the 95% percentile of AgeAccelGrim corresponds to −7.5 years and +8.3 years. A person in the top 95^(th) percentile of AgeAccelGrim (=8.3 years) faces a hazard of death that is twice that of the average person in their straturm (whose AgeAccelGrim equals 0). Specifically, fast aging status is associated with a hazard ratio of HR=2.2=1·10^(8.3). Conversely, a slow ager in the bottom 5^(th) percentile (−7.5 years) faces a hazard of death that is half that of the average person in their stratum, HR=0.49=1·10^(7.5).

DNAm GrimAge Versus Single Stage Estimators of Lifespan

DNAm GrimAge was built using a novel two-stage approach that critically depended on the development of DNAm-based surrogate biomarkers. To justify the utility of this indirect approach, we compared DNAm GrimAge with several DNAm-based lifespan predictors that were developed by directly regressing lifespan on DNAm data (referred to as single stage mortality predictors). To this end, we developed a new mortality predictor, DNAm Mortality (in year units) by directly regressing time-to-death (due to all-cause mortality) on CpGs in the FHS training data. DNAm Mortality was calculated as linear combination of 59 CpGs. The direct approach entailed the constructions of DNAm Mortality, an ElasticNet Cox regression model, and linear transformation of the mortality risk to ensure that the values of DNAm Mortality are in units of years (Methods). Further, we also evaluated the published mortality predictor from Zhang [4] which, remarkably, is based on only 10 CpGs (Methods). The latter two (single-stage) lifespan predictors are highly correlated with each other (r=0.77 in the FHS test data).

The novel age-adjusted DNAm Mortality estimator (HR=1.07, P=3.0E-44) and both versions of Zhang's mortality risk estimator (continuous HR=1.02, P=4.2E-39) lead to a less significant meta-analysis P-value for lifespan prediction than AgeAccelGrim (HR=1.10, P=2.0E-75). AgeAccelGrim also stands out in terms of its meta-analysis P-value for predicting time-to-CHD (AgeAccelGrim P=1.1E-20, AgeAccelMortality P=1.8E-11, AgeAccelZhang P=1.9E-10).

Because smoking is a major risk factor, it is useful to characterize the different lifespan predictors in terms of their correlation with DNAm pack-years. Age-adjusted DNAm pack-years exhibits positive correlations with both DNAm Mortality and Zhang's mortality predictor (r≥0.55). The connection of single stage mortality predictors to smoking can also be observed at the CpG level. DNAm Mortality, Zhang's mortality predictor, and DNAm pack-years explicitly use CpG cg05575921 (in the AHRR gene on chromosome 5p15.33), which has previously been highlighted by epigenome-wide association studies of cumulative smoking exposure [4, 19]. Overall, these results suggest that the two single-stage lifespan predictors relate more strongly to cumulative smoking exposure than AgeAccelGrim.

Association with Blood Cell Composition

DNAm data allow one to estimate several abundance measures of blood cells as described in (Methods)[20, 21]. We previously showed that DNAm biomarkers of aging, which capture age-related changes in blood cell composition are better predictors of lifespan than those that are independent of blood cell counts [9]. Therefore, we hypothesized that several of our novel DNAm biomarkers would exhibit significant correlations with these imputed measures of blood cell composition. This is indeed the case as can be seen from our large-scale meta-analysis across the validation data. AgeAccelGrim is significantly associated with a decrease in naive CD8+ T cells (r=−0.22, P=9.2E-62), CD4+T cells (r=−0.21, P=1.8E-57), and B cells (r=−0.18, P=9.7E-43) and with an increase in granulocytes/neutrophils (r=0.24, P=1.5E-74) and plasma blasts (r=0.22, P=7.3E-63). While these results demonstrate that AgeAccelGrim is associated with an age-related decline in immune system functioning, our cross-sectional analysis does not allow us to dissect cause-and-effect relationships.

Age-adjusted DNAm TIMP-1 exhibits the most significant correlations with the measures of blood cell composition (e.g. proportion of granulocytes r=0.36, P=2.7E-172) followed by age adjusted DNAm Cystatin C (proportion of CD4+ T cells counts r=−0.33, P=3.4E-142). Although many of our DNAm biomarkers are correlated with blood cell counts, this does not mean that these measures only capture changes in blood cell composition. This point is illustrated by the finding that measures of blood cell composition only correlate weakly with our age-adjusted DNAm surrogate markers of smoking pack-years (strongest correlation r=−0.14) and PAI-1 levels (strongest correlation r=0.17) even though both biomarkers are strongly associated with mortality risk and age-related conditions as shown above.

Association with Leucocyte Telomere Length

Leukocyte telomere length (LTL) has been found to be predictive of mortality and cardiovascular disease.

Our meta-analysis reveals a statistically significant but weak negative correlation between LTL and AgeAccelGrim (r=−0.12 and meta P=3.3E-10) across data from the FHS, WHI (BA23 sub-study) and JHS (total N=2,702, 27% White and 73% African American). Similarly, LTL exhibits (weak) negative correlations with our DNAm based surrogate biomarkers for GDF-15 (r=−0.10, meta P=3.4E-7), DNAm PAI-1 (r=−0.10, meta P=5.1E-8) and smoking pack-years (r=−0.09 and meta P=2.9E-6).

Diet, Education, and Life Style Factors

Several previous measures of epigenetic age acceleration in blood have been shown to exhibit statistically significant but weak correlations with lifestyle factors and biomarkers of metabolic syndrome [3, 22]. Here we revisited these cross-sectional studies in the WHI (comprising approximately 4000 postmenopausal women, Methods) with our novel measures of AgeAccelGrim and its underlying DNAm-based surrogate biomarkers (FIG. 6).

All of our (age-adjusted) DNAm-based biomarkers correlate with plasma biomarkers measuring vegetable consumption, but AgeAccelGrim (robust correlation coefficient r=−0.26, P=9E-39, FIG. 6) and DNAm PAI-1 (r=−0.25, P=7E-36) stand out in terms of their strong relationship with mean carotenoid levels (FIG. 6). Similar, but far less significant negative associations, could be observed for self-reported measures of fruit-, vegetable-, and dairy intake. The following results were unexpected because they had not been observed with previous DNAm-based biomarkers of aging: (self-reported) proportion of carbohydrate consumption was associated with lower AgeAccelGrim (robust correlation r=−0.12, P=4E-13) and DNAm PAI-1 (r=−0.15, P=3E-20). Conversely, an increased proportion of fat intake (but not protein intake) was associated with increased AgeAccelGrim (r=0.09, P=2E-8) and DNAm PAI-1 (r=0.13, P=1E-14). Measures of lipid metabolism, triglyceride levels and HDL cholesterol levels, were significantly correlated with AgeAccelGrim (r=0.11 and r=−0.10, respectively) and even more so with (age adjusted) DNAm PAI-1 levels (r=0.34 and r=−0.11). Similarly, measures of glucose metabolism, insulin- and glucose levels, exhibited positive correlations with AgeAccelGrim (r=0.16 and r=0.12, respectively) and with (age adjusted) DNAm PAI levels (r=0.30 and r=0.22).

Similar to what we observed with previous DNAm based biomarkers of aging, plasma C-reactive protein levels exhibited comparatively strong positive correlations with DNAm-based biomarkers, particularly AgeAccelGrim (r=0.28, P=2E-52), DNAm TIMP-1 (r=0.27, P=2E-49), and DNAm PAI-1 (r=0.26, P=1E-46).

Measures of adiposity, BMI and waist-to-hip ratio, are associated with increased AgeAccelGrim, age-adjusted DNAm PAI-1, and other DNAm based surrogate biomarkers. Higher education and income are associated with lower AgeAccelGrim (P=2E-9 and P=2E-6). AgeAccelGrim stands out when it comes to detecting a beneficial effect of physical exercise (r=−0.10, P=3E-10).

Several of our results in the WHI could be replicated in a smaller data (N<625 individuals) from the FHS test dataset that included lipid and metabolic biomarker data. In the FHS, hemoglobin A1C and albumin levels (in urine) exhibited significant positive correlations with AgeAccelGrim, age-adjusted DNAm PAI-1 (0.10≤r≤50.12 and 1.4E-07≤P≤2.3E-03), and to a lesser extent with our other DNAm based surrogate biomarkers.

DNAm-based biomarkers are strong predictors of aging [10, 23]. Several recent articles describe DNAm-based biomarkers for measuring tissue age and for predicting lifespan. This work shows that DNAm GrimAge, which in essence is as a linear combination of age, sex, and surrogate biomarkers for plasma proteins and smoking pack-years, outperforms all other DNAm-based predictors of lifespan and CHD. Moreover, the DNAm-based biomarkers of smoking and PAI-1 might be used independently of DNAm GrimAge to test a host of hypotheses linking smoking and PAI-1 to aging and its related diseases. The DNAm based surrogate biomarker of smoking might complement self-reported assessments of pack-years. The surprising fact that DNAm pack-years outperforms self-reported pack-years in predicting lifespan could reflect erroneous self-reporting. Alternatively, DNAm pack-years may capture intrinsic variation across individuals in the lasting biological damage that results from smoking, i.e., inter-individual sensitivities to smoking.

Notably, only a small fraction of plasma proteins (7 out of 88) could be imputed in this work based on DNAm. In the FHS data, the measurement of the plasma proteins (exam 7) preceded the measurement of blood DNAm data (exam 8) by 6.6 years, suggesting that DNAm is not just a snapshot of the status of these proteins at the time of blood collection. That said, the elucidation of cause-and-effect relationships between plasma proteins and DNAm will require future longitudinal cohort studies and mechanistic evaluations. Our DNAm-based surrogate biomarkers of plasma protein levels may be leveraged by researchers who rely on bio-banked DNA samples without the availability of plasma samples.

Strong evidence supports links between plasma proteins used in the construction of GrimAge and various age-related conditions: ADM levels are increased in individuals with hypertension and heart failure[24]. Plasma B2M is a clinical biomarker associated with cardiovascular disease, kidney function, and inflammation [25]. Plasma cystatin-C is used to assess kidney function [26]. Overall, ADM, B2M, cystatin C, and leptin relate to many age-related traits including cognitive functioning [27-29]. GDF-15 is involved in age-related mitochondrial dysfunction [29]. PAI-1 plays a central role in a number of age-related subclinical and clinical conditions [30], and recent genetic studies link PAI1 to lifespan [31]. The tissue inhibitor of metalloproteinases, TIMP-1, plays an anti-apoptotic function [32].

While AgeAccelGrim stands out when it comes to lifespan prediction and prediction of time-to-CHD, our (age-adjusted), DNAm surrogate marker of PAI-1 level stands out when it comes to associations with type 2 diabetes status, glucose-, insulin-, triglyceride levels and measures of adiposity (body mass index and waist-to-hip ratio).

Inflammation and metabolic conditions are associated with AgeAccelGrim, age-adjusted DNAm PAI1 and age-adjusted DNAm TIMP-1.

Despite their obvious strengths, DNAm-based biomarkers are unlikely to replace existing clinical indices, e.g., blood glucose, blood lipids, blood pressure, etc., in medical practice. Rather, these biomarkers might complement existing clinical biomarkers when evaluating the individual's overall ‘aging’ status. Since DNAm captures important properties of the DNA molecule, these DNAm biomarkers are proximal to innate aging processes [10].

Finally, beyond lifespan prediction, AgeAccelGrim (and several of its underlying surrogate biomarkers) relate to most age-related conditions (metabolic syndrome, comorbidity, markers of inflammation such as C-reactive protein levels) in the expected way, i.e., increased values are associated with deleterious effect. Similarly, higher values of AgeAccelGrim (and several DNAm-based surrogate markers) are associated with a blood cell composition that is indicative of older individuals and shorter telomere length. While the reported associations are statistically significant, the magnitude of the underlying correlations is relatively small in keeping with the concept that aging is multi-factorial and perhaps the most complex of all human traits.

Methods Study Cohort

To establish DNAm based estimators and DNAm GrimAge, we used 2,356 individuals composed of 888 pedigrees from the FHS cohort [16], a large-scale longitudinal study started in 1948, initially investigating risk factors for cardiovascular disease (CVD). The FHS cohort contains medical history and measurements, immunoassays at exam 7, and blood DNA methylation profiling at exam 8. The technology of immunoassay was based on Luminex xMAP assay, an extension of the enzyme-linked immunosorbent assay (ELISA) performed with multiple analyte-specific capture antibodies bound to a set of fluorescent beads. The DNA methylation profiling was based on the Illumina Infinium HumanMethylation450K BeadChip.

We assigned 70% pedigrees (1731 individuals/622 pedigrees) to the training process and the remaining 30% of pedigrees (625 individuals/266 pedigrees) to the FHS test data (Table 3). The training dataset was used to build the DNAm based surrogate markers for plasma proteins, smoking pack-years, and the composite biomarker DNAm GrimAge.

Validation Data from 5 Cohorts

Our validation analyses involved 7,375 Illumina arrays measuring blood methylation levels in N=6,935 individuals from five independent cohorts: the FHS test dataset (N=625), WHI BA23 (N=2107), WHI EMPC study (N=1972), JHS (N=1747), and InChianti (N=924 from 1 to 2 longitudinal measures on 484 individuals, Table 2). All the statistical analyses were adjusted for the correlation structure due to pedigree effects or repeated measurements as described below.

Estimation of Surrogate DNAm Based Biomarkers

We developed estimators for plasma proteins based on blood methylation data. We leveraged immunoassay measurements in the FHS which profiled 88 plasma protein biomarkers (in units of pg/mL), including cardiovascular disease related plasma proteins such as C-reactive protein[33] and growth differentiation factor 15 (GDF-15)[34]. For each protein marker, missing values were imputed by the respective median value. The median missing rate was <0.3%. Next the resulting observed plasma levels were regressed on DNAm data in the FHS training data.

Each plasma protein was regressed on the CpGs using the elastic net regression model implemented in the R package glmnet. Ten-fold cross validation was performed in the FHS training data to specify the underlying tuning parameter A.

We required the predicted variable associated with the target variable with ≥0.35 correlation in both training and test datasets. Only 12 out of 88 proteins exhibited a correlation greater than 0.35 between observed plasma levels and their respective DNAm based estimators in the FHS test data (Table 1). The missing rates of the 12 ImmunoAssay proteins were less than 0.7%. The correlation estimates have a distribution of 0.64±0.12 [0.43, 0.86] (mean±SD [range]) in the training dataset and a distribution of 0.43±0.09 [0.35, 0.66] in the test dataset.

DNA Methylation Data

The Illumina 450K Infinium array profiled almost 486k CpG markers across the whole genome. Methylation beta values were generated using the Bioconductor minfi package with background correction. Our analysis focused on the subset of 450,161 CpGs that are also available on the Illumina EPIC array.

Smoking Pack-Years

The variable “smoking pack-years” attempts to measure the cumulative amount of cigarettes consumed by the smoker. It is calculated by the number of packs of cigarettes smoked per day multiplied by the number of years the person smoked. We computed smoking pack-years using the information up to exam 8 in the FHS cohort.

Definition of DNAm GrimAge

We again used an elastic net Cox regression model[35] to regress time-to-death (due to all-cause mortality) since exam 7 on the 12 DNAm based surrogate markers for plasma proteins and on DNAm PACKYR, chronological age, and sex. In the training dataset, we performed 10-fold cross validation to specify the value of the tuning parameter λ.

Linearly Transform DNAm GrimAge into Units of Years

The final ElasticNet Cox model listed in Table 4 results in an uncalibrated DNAm GrimAge estimate, which can be interpreted as the linear combination of the covariates, X^(T)β, or alternatively as the logarithm of the hazard ratio, log[h(t)/h₀(t)]=X^(T)β, where h₀(t) is the baseline hazard at time. The linear combination, X^(T)β, can be interpreted as an uncalibrated version of DNAm GrimAge. To facilitate an intuitive interpretation as a physiological age estimator, we linearly transformed it so that the resulting estimate would be in units of years. Toward this end, we imposed the following requirement:

the mean and variance of the resulting value, DNAm GrimAge, should be the same as the mean and variance of the age variable in the FHS training data (exam 7). This resulted in the following transformation

DNAm GrimAge=−50.28483+8.3268*X^(T)β,

Observed DNAm GrimAge Model

Our finalized DNAm GrimAge is composed of 7 DNAm proteins, DNAm pack-years, age and gender, yielding DNAm based age acceleration measure: AgeAccelGrim (in units of year). We fitted a Cox regression model based on the observed proteins, self-report pack-years, age, and gender, using the FHS training dataset. This yielded the observed value of GrimAge. We computed age acceleration measures based on the observed and DNAm based GrimAge and compared their performances as risk factors for mortality, using the FHS training and test datasets, respectively.

Statistical Models Used in Validation Analysis

Validation analysis was performed on 7,735 observations across 6,395 individuals (Table 2) coming from five datasets: the FHS test dataset (N=625), WHI BA23 (N=2107), WHI EMPC (N=1972), Jackson Heart Study (JHS, N=1747), and InChianti study (N=924 from 1 to 2 longitudinal measures on 484 individuals, Table 2). Our validation analysis involved i) Cox regression for time to death, for time-to-CHD, and for time to coronary heart failure, ii) linear regression for our DNAm based measures (independent variable) associated with and number of age-related conditions (dependent variable) and physical function score, respectively, iii) linear regression for age at menopause (independent variable) associated with our DNAm measure, iv) logistic regression analysis for estimating the odds ratios of our DNAm based measure associated with any cancer, hypertension, type 2 diabetes, emphysema, and disease free status. The variable of “number of age-related conditions” includes arthritis, cataract, cancer, CHD, CHF, emphysema, glaucoma, lipid condition, osteoporosis, type 2 diabetes, etc. In our validation analysis, we used AgeAccelGrim (the age-adjusted measure of DNAm GrimAge), and used the scaled measures of seven DNAm surrogates for plasma proteins based on the mean and standard deviation (SD) of the FHS training dataset such that the effect size was approximately corresponding to one SD. All the models were adjusted for age, and adjusted for batch effect as needed. To avoid the bias due to familial correlations from pedigrees in the FHS cohort or the intra subject correlations from the repeated measures, we accounted for the correlations accordingly in all the analyses in the following. In Cox regression analysis, we used robust standard errors, the Huber sandwich estimator, implemented in R coxph function. We used linear mixed models with a random intercept term, implemented in lme R function. We used generalized estimation equation models (GEE), implemented in R gee function, for our logistic regression models. Additional covariates related to demographic characteristics, psychosocial behaviors and clinical covariates were adjusted in multivariate Cox models analysis. The additional covariates include BMI (category), education attainment (category), alcohol consumption (gram/day), self-report smoking pack-years, three medical covariates: status of cancer, hypertension and type 2 diabetes at baseline. The categories associated with BMI ranges are a) 18.5-25 (normal), b) 25 to 30 (over), and c) >30 (obese). The categories associated with education attainment are a) less than high school, b) high school degree, c) some college, and d) college degree and above. Both smoking pack-years and education variables were not available in the JHS cohort. Smoking category (never, former and current) was used in the analysis using the JHS cohort.

Meta-Analysis

Unless specified, we used fixed effect models weighted by inverse variance to combine the results across validation study sets into a single estimate by using the metafor R function. A few statistics were combined via Stouffer's method due to different scaling of effect sizes across study sets, such as number of age-related conditions, disease free status and physical function scores.

Heritability Analysis

We conducted heritability analysis using the polygenic models defined in SOLAR [36] to estimate narrow sense h². The robust polygenic model (with t-dist option) was used to estimate heritability of AgeAccelGrim and DNAm based proteins. Heritability is defined as the total proportion of phenotypic variance attributable to genetic variation in the polygenic model. All the analysis were adjusted for age (except AgeAccelGrim) and gender and were performed in R solarius [37].

Two Stage Estimate of Mortality Versus a Single Stage Estimate of Mortality

To develop our single stage mortality estimator, DNAm Mortality, we used ElasticNet Cox regression to regress time-to-death on the CpG markers, chronological age and sex in the FHS training data. We used the same options in the training process (i.e., 10-fold cross validation for choosing the lambda tuning parameter). The final estimator for DNA Mortality is a linear combination of 59 CpGs and chronological age. We used the same method for calibration as we did for DNAm GrimAge and to arrive at an estimate in units of years (DNAm Mortality). A secondary analysis was to compare with the two estimators of mortality developed by Zhang (on the basis of 10 CpGs)[4]. One estimator is a composite score of these 10 CpGs with weights determined by Cox regression with lasso penalty. Using the same method, we calibrated this estimate in units of year (arriving at DNAmZhang) in order to ensure a fair comparison with our other predictors of lifespan. The other Zhang estimator was defined as the total scores of the 10 CpGs with aberrant methylation[4], referred as DNAmZhangScore (range from 0 to 10). Of the 10 CpGs, cg06126421 and cg23665802 were absent in the JHS cohort and were replaced by the medians based on the FHS training data.

AgeAccelGrim Versus Blood Cell Composition

The imputed blood cell abundance measures were related to DNAm Grim Age models using the validation study sets: FHS test, WHI BA23, JHS, and InChianti, involving n=6,003 individuals. The following imputed blood cell counts were analyzed: B cell, naïve CD4+ T, CD4+ T, naïve CD8+ T, CD8+ T, exhausted cytotoxic CD8+ T cells (defined as CD8 positive CD28 negative CD45R negative), plasma blasts, natural killer cells, monocytes, and granulocytes. The blood cell composition imputation of the naive T cells, exhausted T cells, and plasma blasts was based on the Horvath method [38]. The remaining cell types were imputed using the Houseman method [21]. To avoid confounding by age, we used AgeAccelGrim and adjusted all DNAm based surrogate biomarkers by chronological age (by forming residuals). The correlation results were combined across studies via the same fixed effect models.

Diet, Exercise, Education, and Lifestyle Factors

We performed a robust correlation analysis (biweight midcorrelation, bicor [39]) between 1) AgeAccelGrim and its eight age-adjusted components and 2) 38 variables including 12 self-reported dietary variables, behavioral variables, 9 dietary biomarkers, 12 variables related to metabolic related traits and central adiposity, and 5 life style factors. We combined the individuals from the WHI BA23 and WHI EMPC, up to ˜4000 postmenopausal women. The bicor analysis was conducted in the whole sample and in the three groups stratified by ethnicity. Blood biomarkers were measured from fasting plasma collected at baseline. Food groups and nutrients are inclusive, including all types and all preparation methods, e.g. folic acid includes synthetic and natural, dairy includes cheese and all types of milk. The individual variables are explained in [22].

EXAMPLE 1 REFERENCES

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EXAMPLE 2: DATASETS AND MATERIALS OF THE INVENTION Note 1: Description of Datasets

Our study involved four cohorts: the Framingham Heart Study (FHS) offspring cohort, the Women's Health Initiative (WHI), Jackson Heart Study (JHS), and Invecchiare in Chianti, aging in the Chianti area (InChianti). We established our DNAm GrimAge model using the individuals from the FHS cohort. Of the FHS cohort, 1731 individuals were used for the training process in establishing the DNAm GrimAge model and 625 individuals were used for the test process at stage 1. Validation analysis was performed on 7,735 observations across 6,395 individuals coming from five independent datasets: the FHS test dataset (N=625), WHI BA23 (N=2107), WHI EMPC (N=1972), JHS (N=1747), and InChianti study (N=924 from 1 to 2 longitudinal measures on 484 individuals). Below we describe each study cohort/datasets in more detail.

Framingham Heart Study Cohort

The FHS cohort¹ is a large-scale longitudinal study started in 1948, initially investigating the common factors of characteristics that contribute to cardiovascular disease (CVD), https://www.framinghamheartstudy.org/index.php. The study at first enrolled participants living in the town of Framingham, Mass., who were free of overt symptoms of CVD, heart attack or stroke at enrollment. In 1971, the study started FHS Offspring Cohort to enroll a second generation of the original participants' adult children and their spouses (n=5124) for conducting similar examinations². Participants from the FHS Offspring Cohort were eligible for our study if they attended both the seventh and eighth examination cycles and consented to having their molecular data used for study. We used the 2,356 participants from the group of Health/Medical/Biomedical (IRB, MDS) consent and available for both Immunoassay array DNA methylation array data. The FHS data are available in dbGaP (accession number: phs000363.v16.p10 and phs000724.v2.p9).

We computed the total of number age-related conditions based on dyslipidemia, hypertension, cardiovascular disease (including coronary heart disease [CHD] or congestive heart failure [CHF]), type 2 diabetes, cancer and arthritis. Time to CHD or time to CHF was truncated at zero if it occurred before exam 8. Deaths among the FHS participants that occurred prior to Jan. 1, 2013 were ascertained using multiple strategies, including routine contact with participants for health history updates, surveillance at the local hospital and in obituaries of the local newspaper, and queries to the National Death Index. Death certificates, hospital and nursing home records prior to death, and autopsy reports were requested. When cause of death was undeterminable, the next of kin were interviewed. The date and cause of death were reviewed by an endpoint panel of 3 investigators.

DNA methylation quantification Peripheral blood samples were collected at the 8^(th) examination. Genomic DNA was extracted from buffy coat using the Gentra Puregene DNA extraction kit (Qiagen) and bisulfite converted using EZ DNA Methylation kit (Zymo Research Corporation). DNA methylation quantification was conducted in two laboratory batches using the Illumina Infinium HumanMethylation450 array (Illumina). Methylation beta values were generated using the Bioconductor minfi package with Noob background correction³.

Women's Health Initiative

The WHI is a national study that enrolled postmenopausal women aged 50-79 years into the clinical trials (CT) or observational study (OS) cohorts between 1993 and 1998^(4.5). We included 4,079 WHI participants with available phenotype and DNA methylation array data: 2,107 women from “Broad Agency Award 23” (WHI BA23) and 1,972 women from “Epigenetic Mechanisms of PM-Mediated CVD Risk” (WHI EMPC). WHI BA23 focuses on identifying miRNA and genomic biomarkers of coronary heart disease (CHD), integrating the biomarkers into diagnostic and prognostic predictors of CHD and other related phenotypes, and other objectives can be found in https://www.whi.org/researchers/data/WHIStudies/StudySites/BA23/Pages/home.aspx. WHI EMPC is a study of epigenetic mechanisms underlying associations between ambient particulate matter (PM) air pollution and cardiovascular disease⁶. WHI EMPC and BA23 span three WHI sub-cohorts including GARNET, WHIMS and SHARe. 936 EMPC participants were not in any of the WHI GWAS (either GARNET, WHIMS, SHARe, MOPMAP, HIPFX, or GECCO). The largest overlap was with SHARE & GARNET. There was almost no overlap with WHIMS & MOPMAP.

The total number of age-related conditions was based on Alzheimer's disease, amyotrophic lateral sclerosis, arthritis, cancer, cataract, CVD, glaucoma, emphysema, hypertension, and osteoporosis.

DNA methylation quantification for BA23 In brief, bisulfite conversion using the Zymo EZ DNA Methylation Kit (Zymo Research, Orange, Calif., USA) as well as subsequent hybridization of the HumanMethylation450k Bead Chip (Illumina, San Diego, Calif.), and scanning (iScan, Illumina) were performed according to the manufacturers protocols by applying standard settings. DNA methylation levels (β values) were determined by calculating the ratio of intensities between methylated (signal A) and un-methylated (signal B) sites. Specifically, the β value was calculated from the intensity of the methylated (M corresponding to signal A) and un-methylated (U corresponding to signal B) sites, as the ratio of fluorescent signals β=Max(M,0)/[Max(M,0)+Max(U,0)+100]. Thus, β values range from 0 (completely un-methylated) to 1 (completely methylated).

DNA methylation quantification for WHI EMPC Illumina Infinium HumanMethylation450 BeadChip data from the Northwestern University Genomics Core Facility for WHI EMPC participants sampled in stages 1a, 1b, and 2 were quality controlled, normalized and batch adjusted. Beta-mixture quantile normalization was implemented using BMIQ⁷ and empirical Bayes methods of batch adjustment for stage and plate were implemented in ComBat⁸.

Lifestyle Factors and Dietary Assessment in the Women's Health Initiative (WHI)

WHI participants completed self-administered questionnaires at baseline which provided personal information on a wide range of topics, including sociodemographic information (age, education, race, income), and current health behaviors (recreational physical activity, tobacco and alcohol exposure, and diet). Participants also visited clinics at baseline where certified Clinical Center staff collected blood specimens and measured anthropometrics (weight, height, hip and waist circumferences) and blood pressures (systolic, diastolic). Body mass index and waist to hip ratio were calculated from these measurements.

Dietary intake was assessed at baseline using the WHI Food Frequency Questionnaire 9. Briefly, participants were asked to report on dietary habits in the past three months, including intake, frequency, and portion sizes of foods or food groups, along with questions concerning topics such as food preparation practices and types of added fats. Nutrient intake levels were then estimated from these responses. For current drinker, we use the threshold of more than one serving equivalent (14 g) within the last 28 days.

Jackson Heart Study

The JHS is a large, population-based observational study evaluating the etiology of cardiovascular, renal, and respiratory diseases among African Americans residing in the three counties (Hinds, Madison, and Rankin) that make up the Jackson, Miss. metropolitan area¹⁰. Data and biologic materials have been collected from 5306 participants, including a nested family cohort of 1,498 members of 264 families. The age at enrollment for the unrelated cohort was 35-84 years; the family cohort included related individuals >21 years old. Participants provided extensive medical and social history, had an array of physical and biochemical measurements and diagnostic procedures, and provided genomic DNA during a baseline examination (2000-2004) and two follow-up examinations (2005-2008 and 2009-2012). The study population is characterized by a high prevalence of diabetes, hypertension, obesity, and related disorders. Annual follow-up interviews and cohort surveillance are ongoing. In our analysis, we used the visits at baseline from 1747 individuals as part of project JHS ancillary study ASN0104, available with both phenotype and DNA methylation array data. Total numbers of age-related conditions were based on hypertension, type 2 diabetes, kidney dysfunction based on ever dialysis, and CVD.

DNA methylation quantification Peripheral blood samples were collected at the baseline. Methylation beta values were generated using the Bioconductor minfi package with Noob background correction³.

Invecchiare in Chianti, Aging in the Chianti Area (InChianti)

The InChianti (Invecchiare in Chianti, aging in the Chianti area) cohort is a representative population-based study of older persons enrolling individuals aged 20 years and older from two areas in the Chianti region of Tuscany, Italy, http://inchiantistudy.net/wp/. One major goal of the study is to translate epidemiological research into geriatric clinical tools, ultimately advancing clinical applications in older persons. Of the cohort, 924 observations from 484 individuals with both phenotype information and DNA methylation data were including in our studies. The observations were collected from baseline in 1998 and the third follow-up visit in 2007. All participants provided written informed consent to participate in this study. The study complied with the Declaration of Helsinki. The Italian National Institute of Research and Care on Aging Institutional Review Board approved the study protocol. We computed the total number of age-related conditions based on cancer, hypertension, myocardial infarction, Parkinson's disease, stroke and type 2 diabetes.

DNA methylation quantification. Genomic DNA was extracted from buffy coat samples using an AutoGen Flex and quantified on a Nanodrop1000 spectrophotometer prior to bisulfite conversion. Genomic DNA was bisulfite converted using Zymo EZ-96 DNA Methylation Kit (Zymo Research Corp., Irvine, Calif.) as per the manufacturer's protocol. CpG methylation status of 485,577 CpG sites was determined using the Illumina Infinium HumanMethylation450 BeadChip (Illumina Inc., San Diego, Calif.) as per the manufacturer's protocol and as previously described¹¹. Initial data analysis was performed using GenomeStudio 2011.1 (Model M Version 1.9.0, Illumina Inc.). Threshold call rate for inclusion of samples was 95%. Quality control of sample handling included comparison of clinically reported sex versus sex of the same samples determined by analysis of methylation levels of CpG sites on the X chromosome¹¹. Methylation beta values were generated using the Bioconductor minfi package with Noob background correction³.

Note 2. DNAm Based Surrogates for Plasma Proteins

The model of DNAm GrimAge is composed of seven DNAm based plasma proteins, DNAm based pack years, age and gender. Below we briefly describe the seven plasma proteins. ADM (adrenomedullin) is a vasodilator peptide hormone. Plasma ADM, initially isolated from adrenal gland, is increased in individuals with hypertension and heart failure¹². A recent study showed that ADM was involved in age-related memory loss in mice and aging human brains¹³.

B2M (Beta-2 microglobulin) is a component of major histocompatibility complex class 1 (MHC I) molecular. Plasma B2M is a clinical biomarker associated with cardiovascular disease, kidney function, inflammation severity¹⁴. B2M is a pro-aging factor associated with cognitive and regenerative function in aging process and suggests B2M may be targeted therapeutically in old age¹⁵. A previous study showed that systemic B2M accumulation in aging blood promoted age-related cognitive dysfunction and impairs mouse models¹⁵.

Cystatin C or cystatin 3 (formerly gamma trace, post-gamma-globulin, or neuroendocrine basic polypeptide) is mainly used as a biomarker of kidney function. Plasma cystatin-C is a clinical relevant biomarker indicating kidney function¹⁶. Cystatin-C seems plays a role in cardiovascular disease¹⁷ or amyloid deposition associated with Alzheimer's disease¹⁸.

GDF-15 (growth differentiation factor 15) is one of transforming growth factor beta subfamily. GDF-15 has been implicated in aging and age-related disorders. It also plays a role in age-related mitochondria dysfunction¹⁹.

Leptin is a hormone predominantly in adipose cells. Leptin plays a role in regulating energy balance by inhibiting hunger and is implicated in Alzheimer's disease²⁰.

Plasminogen activator inhibitor antigen type 1(PAI-1) is the major inhibitor of tissue-type plasminogen activator and unokinase plasminogen activator. PAI-1, released in response to inflammation process, plays a central role in a number of age-related subclinical (i.e., inflammation, atherosclerosis, insulin resistance) and clinical conditions (i.e., obesity, comorbidities)²¹.

TIMP-1 or TIMP metallopeptidase inhibitor 1 is a tissue inhibitor of metalloproteinases. It is also involves chromatin structures, promoting cell proliferation in a wide range of cell types, and may also have an anti-apoptotic function²².

Estimation of Blood Cell Counts Based on DNAm Levels

We estimated blood cell counts using two different software tools. First, Houseman's estimation method²³ was used to estimate the proportions of CD8+ T cells, CD4+ T, natural killer, B cells, and granulocytes (mainly neutrophils). Second, the Horvath blood cell estimation method, implemented in the advanced analysis option of the epigenetic clock software^(24,25), was used to estimate the percentage of exhausted CD8+ T cells (defined as CD28−CD45RA−), the number (count) of naïve CD8+ T cells (defined as CD45RA+CCR7+) and plasma blasts cells. We and others have shown that the estimated blood cell counts have moderately high correlations with corresponding flow cytometric measures^(23,26).

TABLES

TABLE 1 Reproducibility and age correlations of DNAm based surrogate biomarkers Training Test (N = 1731) (N = 625) Observed Observed Correlation (r) biomarker Age biomarker Age DNAm based surrogate adrenomedullin 0.65 0.63 0.38 0.64 beta-2-microglobulin 0.62 0.83 0.43 0.85 CD56 0.86 0.17 0.36 0.17 ceruloplasmin 0.56 0.04 0.49 −0.02 cystatin-C 0.58 0.81 0.39 0.83 EGF fibulin-like ECM protein1 0.59 0.72 0.41 0.87 growth differentiation factor 15 0.74 0.71 0.53 0.81 leptin 0.68 0.06 0.35 0.05 myoglobin 0.50 −0.04 0.38 0.03 plasminogen activator 0.69 0.19 0.36 0.16 inhibitor 1 serum paraoxonase/ 0.57 −0.22 0.51 −0.22 arylesterase 1 tissue Inhibitor 0.43 0.92 0.35 0.90 Metalloproteinases 1 smoking pack-years 0.79 0.17 0.66 0.13

Legend

This table reports the correlation coefficients between the observed marker (i.e. observed plasma protein level or self-reported smoking pack-years) and its respective DNAm based surrogate marker in 1) the FHS training data and 2) the FHS test data. Each of the DNA based surrogate biomarkers (rows) leads to a correlation r≥0.35 in both training and test datasets (columns 2 and 4). DNAm based pack-years is highly correlated with the self-report pack-years in both training and test datasets (r≥0.66). The table also reports the correlation coefficients between the DNAm based surrogate biomarkers (rows) and chronological age in the FHS training and test data (columns 3 and 5).

TABLE 2 Overview of the cohorts used in the validation analysis Years of Smoking status Study N Female Age Never Former Current Pack-years Follow-up FHS*  625  53% 66.9 ± 8.64 37% 52% 10% 14.7 ± 19.91 7.7 ± 1.78 test [61, 73] [0, 23] [7.3, 8.8] WHI BA23 2107 100% 65.3:17.1 52% 36% 10% 9.5 ± 18.55 16.97 ± 463 [60, 70.9] [0, 12.5] [15.8, 19.9] WHI EMPC 1972 100% 63.3 ± 7.03 52% 38%  9% 9 ± 17.27 18 ± 4.02 [57.9, 68.7] [0, 12.5] [17.9, 20.1] JHS 1747  63% 56.2 ± 12.31 65% 21% 14% NA 11.7 ± 2.55 [46.5, 65.4] [11.2, 13.1] InChianti**  924  54% 67 ± 16.64 57% 29% 14% 10.3 ± 17.33 5.4 ± 4.84 (484) [60, 78] [0, 16.8] [0.1, 9.3] NA = not available. Quantitative variables are presented in the format of mean ± SD [25^(th), 75^(th)]. *The distribution of age is based on exam 8. **The statistics are based on the number of 924 observations across 484 individuals.

Legend

This table summarizes the characteristics of 6,935 individuals (corresponding to 7,375 Illumina arrays) from five independent cohorts that were used in our validation analysis. For example, up to two longitudinal measurements were available for each of 484 individuals in the InChianti cohort.

TABLE 3 Characteristics of the Framingham Heart Study Description of the Framingham Heart Study (FHS) Offspring Cohort that was used to train and test DNAm GrimAge. We assigned 70% of pedigrees to the training dataset and the remaining 30% pedigrees to the test dataset. Training Test N = 1731 N = 625 N_(pedigree) 622    266    Female  54%  53% Smoking Never  41%  37% Former  51%  52% Current  8%  10% Age exam 7 59.6 ± 9.05 [53;67] 60.3 ± 8.59 [54;66] exam 8 66.2 ± 9.08 [59;73] 66.9 ± 8.64 [61;73] PACK years 13.8 ± 20.17 [0;22] 14.7 ± 19.91 [0;23] Follow-up (years) since exam 7 14.5 ± 1.69 [14;15.6] 14.4 ± 1.82 [13.9;15.6] since exam 8 7.9 ± 1.65 [7.5;8.9] 7.7 ± 1.78 [7.3;8.8] Quantitative variables are presented in the format of mean±one standard deviation, SD and the interval: [25^(th), 75th] percentile. Immunoassay array was profiled at exam 7; DNA methylation array was profiled at exam 8.

TABLE 4 Multivariate regression model for estimating DNA GrimAge Coefficient values for computing the uncalibrated version of DNAm GrimAge based on the underlying covariates (rows): chronological age, sex (an indicator of female), and eight DNA methylation (DNAm) based variables (rows). The columns report the name of the covariate (e.g. DNAm based biomarker), its abbreviation, regression coefficient, and the number of underlying CpGs that underlie the surrogate biomarker (defined in stage 1). As noted, this model yields uncalibrated DNAm GrimAge. The finalized DNAm GrimAge is based on transforming the raw variable into a distribution in units of year (see Methods). Number Covariate Abbreviation Coefficients of CpCs DNAm adrenomedullin DNAm ADM 0.007903 186 DNAm beta-2-microglobulin DNAm B2M 4.59E−7 91 DNAm cystatin-C DNAm Cystatin C  3.5E−6 87 DNAm growth differentiation factor 15 DNAm GDF-15 0.000349 137 DNAm leptin DNAm Leptin −7.3E−6 187 DNAm plasminogen activator inhibitor 1 DNAm PAI-1 2.56E−5 211 DNAm tissue inhibitor metalloproteinases 1 DNAm TIMP-1 0.000144 42 DNAm pack years DNAm PACKYRS 0.030398 172 Chronological age Age 0.030082 — Female Female −0.22847 —

TABLE 5 Comparing self-reported versus DNAm based estimates of smoking pack years predicting time-to-death. Two Cox regression models for time-to-death (due to all-cause mortality) were used to evaluate the predictive power of self-reported smoking pack years and its DNAm based surrogate biomarker, respectively. The survival analysis was conducted across five datasets from four independent study cohorts as detailed in the first column). The column report the variables, the sample size, the tota number of deaths during the follow-up period, the hazard ratio associated with a 1 unit increase in the variable, and the corresponding Cox regression p-value. To compare the performance of the two variables, one should focus on the p-value as opposed to the hazard ratio (because the latter depends on the distribution of the underlying variable). Surprisingly, this comparative analysis indicates that the DNAm based surrogate biomarker of smoking pack years leads to a more significant (smaller) Cox regression p-value than that of the self-reported variable. Data Pack years N # death HR P FHS train DNAm surrogate 1729 219 1.04 8.10E−23 Self-reported 1729 219 1.02 1.12E−11 FHS test DNAm surrogate 625 88 1.04 8.51E−5  Self-reported 625 88 1.01 2.13E−3  WHI BA23 DNAm surrogate 2107 765 1.02 7.17E−12 Self-reported 2029 734 1.01 7.03E−12 WHI EMPC DNAm surrogate 1972 505 1.03  2.1E−19 Self-reported 1904 484 1.02  6.4E−19 InChianti DNAm surrogate 924 209 1.02 5.33E−4  Self-reported 924 209 1.01 1.78E−1  HR = hazard ratio

TABLE 6 Comparing ImmunoAssay versus DNAm based estimates of plasma proteins wrt. predicting time-to-death. Two Cox regression models for time-to-death (due to all-cause mortality) were used to evaluate the predictive power of our study plasma proteins measured by ImmunoAssay versus its DNAm based surrogate biomarker, respectively. The survival analysis was adjusted for chronological age, center, and family structure, conducted in the FHS training (N/number of death = 1729/219) and test(N/number of death = 625/88) datasets, respectively. For each plasma protein, we list the hazard ratios (HR) and the corresponding Cox regression p-values of the protein levels based on ImmunoAssay measure and DNA methylation estimate, respectively. All the test variables were standardized based on the FHS training dataset such that each HR corresponds to an increase in one-standard deviation. To compare the performance of the two variables, one should focus on the p-value as opposed to the hazard ratio (because the latter depends on the distribution of the underlying variable). The analysis for leptin levels was stratified by gender due to its highly correlation with gender. HR* P-value Protein data ImmunoAssay DNAm ImmunoAssay DNAm Adrenomedullin training 1.23 1.36 4.53E−4  3.85E−7 test 1.33 1.06 2.92E−2  7.23E−1 Beta-2 training 1.31 1.51 7.42E−8  9.87E−8 Microglobulin test 1.22 1.94 133E−2  2.92E−3 Cystatin C training 1.25 1.53 1.32E−4  1.44E−8 test 1.13 1.53 5.40E−2  1.92E−2 GDF-15 training 1.24 1.20 3.82E−5  2.58E−3 test 1.83 1.63 6.86E−13 2.32E−2 Leptin training male 1.28 2.23 1.03E−1  9.12E−6 test male 0.99 1.11 9.74E−1  6.83E−1 training female 0.93 0.97 4.81E−1  8.38E−1 test female 1.15 1.18 3.25E−3  5.91E−1 PAI-1 training 1.14 1.31 3.95E−2  3.63E−6 test 1.19 1.37 7.42E−2  8.69E−4 TIMP-1 training 1.33 2.61 1.38E−5  1.06E−8 test 1.29 3.30 1.71E−2  3.79E−4 *in units of one SD. The p value based on a DNAm variable was marked in bold if it was more significant than the one based on the corresponding ImmunoAssay variable. GDF-15 = growth differentiation factor 15; PAI-1 = plasminogen activation inhibitor 1; TIMP-1 = tissue inhibitor metalloproteinase 1.

TABLE 7 Mortality prediction in the FELS based on DNAm- and observed versions of AgeAccelGrim The fact that AgeAccelGrim is a composite biomarker based on DNAm based surrogate biomarkers of plasma protein levels and smoking pack-years begs the question whether a predictor of lifespan based on observed values, i.e. observed plasma protein levels and self- reported smoking pack-years, outperforms its DNAm based analog? To our surprise, relatively little is gained by using observed values as will be shown in the following. Analogous to our construction of DNAm GrimAge, we used a Cox regression model to regress-time to-death on the observed plasma protein levels and self-reported pack-year in the training data (Methods). As before, the resulting mortality risk estimator (defined as linear combination of the observed biomarkers) was linearly transformed into units of years. The resulting predictor will be denoted as observed GrimAge and its age-adjusted version as observed AgeAccelGrim. The table reports the predictive accuracy for the two age acceleration measures based on observed values versus DNAm based surrogates when it comes to predicting time to death (due to all-cause mortality). The table reports the sample size (N), the number of deaths during the follow up period, the hazard ratio associated with a 1 unit increase in the variable, the Z statistic, and the Cox regression p value. To compare the performance of the two variables, one should focus on the p- value as opposed to the hazard ratio (because the latter depends on the distribution of the underlying variable). Rows correspond to the results in the training data and the test data of the FIB. An unbiased comparison in the test from the HIS reveals that the hazard ratio associated with observed AgeAccelGrim (HR = 1.10, P = 3.2E−7) is similar to that of its DNAm based analog AgeAccelGrim (HR = 1.12, P = 8.6E−5). The results in the training data of the FHS are biased and should be ignored. FHS AgeAccelGrim N # death HR Z P Training Observed 1729 219 1.12 10.54 5.65E−26 (N = 1731) DNAm based 1729 219 1.14 11.85 2.15E−32 Test Observed  625  88 1.10  5.11 3.18E−7 (N = 625) DNAm based  625  88 1.12  3.93 8.55E−5 HR = hazard ratio.

TABLE 8 Heritability analysis of observed and DNAm based variables Robust polygenic model was performed to estimate heritability using the FHS test pedigree datasets. We estimated the heritability of observed and DNAm based AgeAccelGrim and it associated proteins. The observed AgeAccelGrim was defined in manuscript and the observed protein levels were based on ImmunoAssay measures. Additional analysis was applied to other epigenetic measures of age acceleration: age-adjusted DNAm PhenoAge (AgeAccelPheno)²⁸, age-adjusted DNAm age based on Hannum et al. (AgeAccelHannum) ²⁷ and age adjusted DNAm age based on Horvath (AgeAccelResidual)²⁴. Heritability P value Trait ImmunoAssay DNAm ImmunoAssay DNAm AgeAccelGrim 0.37 0.30 6.0E−3 2.2E−2 AgeAccelPheno NA 0.11 NA 2.5E−1 AgeAccelHannum NA 0.19 NA 7.7E−2 AgeAccelerationResidual NA 0.44 NA 1.8E−3 Adrenomedullin 0.42 0.38 3.7E−3 2.9E−3 Beta 2 microglobulin 0.34 0.45 3.3E−3 2.4E−3 Cystatin C 0.32 0.09 2.9E−2 3.1E−1 GDF-15 0.24 0.21 6.0E−2 9.1E−2 Leptin male 0.58 0.31 4.8E−2 1.8E−1 Leptin females 0.34 0.38 1.1E−1 1.3E−1 PAI-1 0.34 0.51 7.1E−3 6.2E−4 TIMP-1 0.31 0.24 2.0E−2 9.7E−2 GDF-15 = growth differentiation factor 15; PAI-1 = plasminogen activation inhibitor; TIMP-1 = tissue inhibitor metalloproteinase 1; NA = not applicable.

TABLE 9 CpG Methylation Sites Associated with one or more physiological factors associated with an age of an individual cg Sequence identifier DNA Sequence ID Number cg20462449 CGGGACCGTTTGGCCCTCTGTGACCCAAGTCAGCCTCGGCGAAGTGCTTT 1 cg19802390 CGCGGCCAGGCAGCGAACCCCACTAGGAGGCACCCCAGGCCCCTGGGCTT 2 cg14084907 CGCGCCTGGGCCCCCAGGACCTCTTGCAGACGGGAGTCCCTCAGACAGTG 3 cg23251761 GGTTGCTTCCTTTTGTGCCCCCCGGGGGTCAGGAATCCCAGGTTTCTCCG 4 cg14825555 CTGAGGCCACCTGGGGCTGGGGATCCCACTCTTCTTGCAGCTGTTGAGCG 5 cg04967775 AGGGCCCCCTGGGCCTGTACCAAGGTCTGCGACAGATGGGTGGGGAGCCG 6 cg05817517 GCCTGGCAGAATCAGGAGCCGGGGGAGTAGGGCTCCCGGAAAGTTTATCG 7 cg00500789 GAGGCAGTGGGCGGGGCTGAAGCTGCAGGTCTGGCAGAGGGCGGGCTGCG 8 cg23800435 GTGGGCGGGGCTGAAGCTGCAGGTCTGGCAGAGGGCGGGCTGCGGCAGCG 9 cg08871545 CGCAAGCATGAAGCGTTTAACTGTGCAACACAAATCAACAACCTGGGCTC 10 cg04135242 CGCACCCACTGAATCAGAAGCTGTAGGAGCGGGAGCCAGTGGTTTGCATT 11 cg25189904 CGCTGCCAAACCCCTACACGAAAGTTTGGTTCTGTTCCAGAAACTATGGA 12 cg16511983 GGACAAAAATCCTCGGTGGCGGGGGCAAAAAGTCGCGGTGACAAAAAGCG 13 cg12067764 TTTGAAGCCCTTATGTCTATGGTAATCTGTTACAGCCCCCGCAGGAAACG 14 cg09396704 CGGGCACCTTGCTGCTGGCCCTCGGCCCCACGCACCTGCCCTTGGCTGCC 15 cg17440248 CGTGAATTGAGATGTCATGCCATTGTAAGATTTTTAAAAAGGGAAGGACA 16 cg01491219 CGGCGCGCACTGCACAATGAACATGCAAAACACCTTCAACTGCACTTGCT 17 cg02356786 CCATAGCGGTGGGCCTTTGGCGACGTCAGAGGCGCGGGTGTTCGCCTACG 18 cg19012696 AGCTCCTGCCGTGGTGGTGAAATATTGTGGACTGCTTGGCTGACTACACG 19 cg17886420 CGTGCTCAGCTGATAAAATGCTAAATAGCATCAATGCTGACCATGCTTCT 20 cg12465010 CGTGTGTACACATGTATGTCTTTCAAGCAAGGCTATGTATTACTCAGAAC 21 cg15777335 GTCAGGGAGATAAGTCCCTTTGCGGAATGTCAAGACGGTGAGCAGGCGCG 22 cg03634479 CGCTTCTTAAGGGAAAGGCATGAGGTGGGACAGGCAAACCTTGGCAAGTG 23 cg13458005 CGGCAGGCATCCCCTCTACCTGACCGGCTTCTTCGGAGGATACATCTTGG 24 cg14371731 ATTAGCGGAGCCTCCGCCTATGATTGGCTTCGCCCGGGAAGCTGGAGACG 25 cg23322172 CGGGGGCTCTTCCTGCTCAAGCTGGGACTTAGCCTGCTCATGTTGTTGGC 26 cg02089135 CTTTTTTTGGCATTGGAATTGGCAAGTGGCTGCCCTTGTCTGTTACAACG 27 cg05747812 CGGGGTGCAGGTCAGGGCTCCAGGTGTCACCTGCCTGTCCACACGGGGCT 28 cg11019008 CGCCGTGTCCCTCACGCTGGCTTTCAGAAGGCTTCACGCAAGTGGGCTCA 29 cg02580250 CGGGTGCTGTTATCACCCCAATTTCACCAATGAGGAAAGAGGTGCAGAGA 30 cg24869272 GTGGTTTTCTACCTGGACCTCTCCTTCATCTTCCTCCTAGAACTGAAGCG 31 cg23938637 GCTGTGGCGTCTCCAACTACACTGACTGGTTCGAGGTGTACAACGCCACG 32 cg09690118 CGTGGACTTCCCATCCCCTGGACCCCACGGCGGGGACAAGGAAACCTACA 33 cg01518025 AGCCTCACTGAGGAAGTGACTGTCCCAGGCTCTAGAGGTGCCTGAGGGCG 34 cg06809342 AGACGGGGAGGCCGGGCAGAGACGCTCCTCACCTCCCAGACAGGGTTGCG 35 cg00161556 CGACCCTTTGTGGAGGGCACTGGAGGAGCTGGCATTTAAGCCCTGTCCAG 36 cg06911753 AATGCGTCGCAGTCAAGACAGCGCAGGGGCCTGGGCTGCCGGGCGCTGCG 37 cg04265051 CAAGGGCACACAGCGAGGCAGTTTCAGGGCGGGCAGCCTGGGGCCCCACG 38 cg16385335 CGGGGCAGTGCTTTCCCAGAAGGATTGCTCGGCATCCTGCCCTTCCCAGA 39 cg11660018 CGCACAGCACTGAGTGCTGTAAGGAAATGCAAAGCATTGGTGGCCCTGCC 40 cg23771366 CGGTGCCTCCAAAGAACAGAAACCTGTATTCCCATGGCAGGGCCACCAAT 41 cg12051762 TCTCAAACTCGGGGCCAGAGAACAGTGAAGTAGGAGCAGCCGTAAGTCCG 42 cg10130564 CGCCCCGGCCCGCTCCATCTCCAAAGCATGCAGAGAATGTCTCGGCAGCC 43 cg19174059 GCACACACAGCAGCAGCACTGCCACCCAGAGCAGGGCTAAGATGAACACG 44 cg04832325 CGGGTATTACTGCCATCGTCGTACCAGCAGGGAAACAAACTGAGACTCCG 45 cg08161922 CGCGGCTCCATACTATTGCCCAGTGTTTTCCGAGCATCTCCATCCTTGGC 46 cg00417288 CAGTCCGTCATCTCATCTGGGGAAACTGTTTGCCTTTTTCTGATACTACG 47 cg21368094 CGTGTGCACATGCGCACTCTCATATGCCACCCCATGTGCCTTTAGTCCAC 48 cg05661164 CAGGAGATGCCGCGGTAGGGGGCGGCGGTGATGCAGCAGCGGCTTGGCCG 49 cg09244872 CGGGTGCCCAGGAGCCCACAAAACGGCCAGAGGAGAAATGCTTTCAAAGG 50 cg22277972 CGGCCTTCTGGCTGGCAAGCCCCTGTGGGCCAGGAAGCTCAGACCTAACC 51 cg13406893 AGTCACTGAATCATGAGTGACGGTAACATAAATGCCCATCGGGGGAAACG 52 cg12384004 ACTCGCCTTGATGTTCATTGGGGTTCAAAATAATCAAGTTCTGCAGAACG 53 cg17116694 GGCCAAGGCAGGCGGCTGGGAGGCGTAGGTTGTAGCGAGCCGAGATCACG 54 cg25939203 ACGATGTTGGCTCACTGCATCCTCCACCTTCTGGGTTCAAGCGATTCTCG 55 cg02922734 GTCATCCCCGGGGAGAGGCTCTCAATGGGGAGTCCCATCTGCAGGCTGCG 56 cg01873886 CGGGTTCCTTTTAAAGTGCTGCTAGCGCGCACTCGCCCTCTCAGCGTTGC 57 cg01637125 CAAGGCCACTGCAATTTTTGGGATCTAGTGAGTCTGTAGGAAATGTAACG 58 cg10920224 TGAGGTCAGGCGTGCCCTTGCGTAGCACAATCGCGGGGAGGGGTGGCTCG 59 cg18425651 GCATGTGGCATGGGTGGGGTGGCCAGCACGCTACAGGGGTTTCCTATGCG 60 cg13316619 CGGAAGAGGCTCCAGTGTCCTCAGCAGACATGCTTTATTTGGTTAAAGCA 61 cg23047825 AAGCTTCAGTGAATGTCCCCCGCTTCTAAGATCCTCTTCTTTTTGTCACG 62 cg08923669 CGCCAGGCTCGCGATGCCTCTACACAAGTATCCCGTGTGGCTCTGGAAGC 63 cg06259664 CGGGAGAACCTTCTGTGCAGGCCCGGCGCTGCTCCACTCCAGGAAAGAAA 64 cg05090127 GAGGCAGGGCAGGCAGACCTGCAGTCACTCCTCTGCAGAGTCGCACACCG 65 cg01960979 CGCAGAGAAGGAAATAACTTGTTCAAAAGAACCTACTGGGGTGTGTGTAC 66 cg06213463 GGGAGTGGGCTGGCAGTGCGCGCACAGCGGCGGCGAGTGGGTCGTGCACG 67 cg01773760 TCGTTCCTCATGAAAACATCTTGAGTCCAGGATCTCCAATTGCACTCACG 68 cg01130056 TTCTCTCAAAACTCATCACCCACTTTTCTTTAACTGTTTGTTATAAGACG 69 cg03974717 CGGACCTAGACTCCAACAGTGACAGCTCTGAGTAAAGAAGTCGCCGAAGC 70 cg26573797 GAAGGAGAAGCACCCGGAGCCGGGGGGCTCCTGTGTCTACTGCAAAAGCG 71 cg06127256 TGGCCGGGATCCCGTGCTTGGCATACAGCCGGTCTTGTTCTCATTGTCCG 72 cg03574306 TTCATGGGTGTGGTAGCTTGTGAGACCGGCCGGCTGCGCCCATGCCCCCG 73 cg08439518 TTTAACCTTTCCAGGCCTCAGTTTCTTCATCTGTTCAGTGGAAATGATCG 74 cg11235787 TAGTTTTCACCACCCTGCCTGGAGCAGCACAGCCAATATTGGCAGACTCG 75 cg20361594 CGCAAGCCGGCCCTCACCCAGTATATTGATAGGGCCATTGAGGGGTTCCG 76 cg13029847 CGGCCTGCTAAGGAACCTCAATTAATAGCTCACTGTAGCCTTCTGATTCT 77 cg00126959 GACCCAGAGGCACTGGGGTAGGGCTCGGCAGGGCGGGTCCGGGCGGAGCG 78 cg24093538 CTGGGCTCCATGTGTAATTTCAGGGCTCATGGCCACACCCAGCATTGACG 79 cg16067628 CGGGTAAGCCCCGCTGTGTTCTCATACCCGAACGGCAGGAACACAGGCAG 80 cg15258649 CGCACAGAGCCGCACGGGACTCAGTTTCCACACACACAGAGCCACACGGG 81 cg03294557 TAAGAATAACTTTTGCCTCCTACTGTGATTCTGAGGCCTCCCCAATCACG 82 cg23777956 GTGGTGACAGAGCCACAGAGGGCTGTGAGCTTGCCCGGCCCCAGGTAACG 83 cg03636183 TGGTGGTGGGGCTGCCGGCCAATGGGCTGGCGCTGTGGGTGCTGGCCACG 84 cg06418475 CGGGGCGCTGAGCTCAGGCCCAGAACTGGCTGATTTCAGGGATACCCAGG 85 cg27152890 CGGCGCAGACACCCCGTTCGGACGATCAGAGAGTGCCCCAACCCTACACC 86 cg09438320 GATGACCGGGATACAGTTTGCTGCATTGCAAGGGCTCACCCAGTGTTCCG 87 cg12157673 GTCAAAGGACAAGCCCCTGGGACCTGCCCAATGCTGGTTCAGGGGAAGCG 88 cg22455450 CGGGGCGGAATATACGATTTCATGCTGATGGCCCACAGAACATAACAGAA 89 cg08980304 CGTGTGGACGCGGGAGGAAACCAGAGTGCCCAGAGACAACCCACGCAGAC 90 cg26881591 CCATGCAGGGCTCTAGACAGCGGCGCTGCAGAGCTTCCTGAGGTCGGCCG 91 cg14528537 CTAGCTTTAGCTGCACATTAGAGTCACCTGCAGCTCTTTTCAAACTGTCG 92 cg04053045 GGGGGGTCAATACTCACCTGGTCGTAGACAGGAGCCCTGGAGTCGGAGCG 93 cg05515143 CGTGTCTCAGTGACGCTGATTCTGCTTCACAACAAAAGCAAAACAAGAAC 94 cg06625640 CGGAAAAGGGAGTGGACAGGAGGGGTACAGAACCAGGCTAATGAGACAAT 95 cg00102512 GCTGGCTTTCAGGATCACTATCCAGCAGCAGCTCCGACTTCCAGATTCCG 96 cg06372850 CGCTCTCTGGCAAATGTAGAGGCTATTTCTGCTTTTTACAAGAGGTGCTG 97 cg16978914 CGGCCCTCCAAAATTGCCCCCACATTGGCTGCCTTATAAATATGTCTGTG 98 cg00706683 CGGGTTCAGTCATTGAAAGCCTTCTTGTAATTGCTGAAGGGTATTTGTTT 99 cg06644428 CCTCTTTCTGCTCGCCGAAGGCCGCGATGGTGCCATAGGTGAGCTTTTCG 100 cg21566642 CGCCTCCTGAGCCTGACTCTGCTCTTCCCGCAGCTTTGGCCCCCCGGGGC 101 cg21120063 CGGGCACGATGTCCCAGGAGGACAGAGTTTGCATTGATGAAAACCCTTCC 102 cg00356999 CGCTGGGTCTGCTCCCATTGGCTCAGGTCATCTGAGCAGCTGTGCTCTGT 103 cg02931526 GGTGCTCTGGACCGGCAGCCATGGGAATGTCCTAGTCGGCAGCAGTGTCG 104 cg19393314 CTCACGTACAGTCTCCTCCCTGTACACGAAGGAGACTCTGCTCTAACACG 105 cg05303999 CGCTGGGATCGTGGCTTATGGCGCAGAGACTTCCCCATGACCAATCATTC 106 cg16416603 CGGGCACCCTGAGGTTGCCCCCATTCTTTGAACAGCTATGGACGGGGACC 107 cg05276137 CGGGCTTCCCGGCAGCAGGTTACTCTTTAACGCTCGACTGCCCACCTCCA 108 cg16983817 CGCCCTGATTGGATAGTAGTGCAGGCACCGCGCCCTCAGGCCTTGCGTGT 109 cg00744433 CGCAACTGCAGTTTAGCCTGGTGAGTTGCAATTCTGGCTGCACATGAAAA 110 cg08706141 AGTTTTACAAGCCCTCACATAAATCTACACAGCTAACATGCACAAGTCCG 111 cg03860256 CGCCTTGCGCGCTCGGCATGACGTCACTCCCACCAGGCACCTCGCCTTGT 112 cg15876825 AGGCGATCCTTGCGCTTGGGCAGACAGCACGTCACGGCTTTTATCTGCCG 113 cg21770393 AGTCAGACGTCAGGACCTGCTGTGGGGATGGACCCACAGCTCATCCCCCG 114 cg01206872 CCCAGTACTTTGGGACGCCAAGGTGGGTGGGTCACAAGGTCAGGAGATCG 115 cg02019988 TACCCCAGCTAGGCCCCTTCCCCGCAGAGGATGTGTTTCTGTTGTCCTCG 116 cg04785972 GAGGTGGGCGAGGCAGGGCCCACAGACATTGGTGCAGGCTCAGCCGTGCG 117 cg19859270 CGACTGGCCACACAATGGCCAGGTAGCGGTCAACACTCATGCAAGTGAGC 118 cg16665506 CTTCCCCTCAGCCTTGGTTTCCATATCTGGAAGATGGGTTACCAATAGCG 119 cg01328703 ATGTCACTTGCACAAGAGCCAGTCATGCCACAGGGCTAGCCTTAATTTCG 120 cg17894318 AAATAACTTGTCCTTTACTTACCCTGCACATCAAATCTTCTCGCCCTGCG 121 cg17500754 ATTTTTATTTTATTCCTGCCATTGTTTCTCCTTCGAGTCAATATCTTCCG 122 cg21924273 GGGCGCCAGCCGGCGAGGAGCGACGGGAGCCTGGGGTGGGTGGCGAGGCG 123 cg22518157 CGGCTGTGGTTTCCTGTGCGTCCCAGGTGAGATCTTGCTCTTTACAGGGT 124 cg22659049 AGGACGACAAATGGCAAGATGTGAGTTGTGGCCAAGGCGCGCGGGCAGCG 125 cg25806704 CGGGCTTAGTAAGCCCCTGGAGAAAGGAATCAAAGATGTTTCTGAGGCCT 126 cg07973162 CGCTGGCCACCCTCCTAAAGGATCACTGTGGTGCCAGGCAGGAATGAGCT 127 cg10144483 CGGTGTGCAAACCTGCAATGAATTGCCCAGTGCCTTCTTGTTTGTAACGT 128 cg11554391 ATTATAGAGAATATTAAGATGCTTTTGGATCCTGAGCTTCCCAGGAAGCG 129 cg05575921 CGGCTGGGTCTCATCTGACACGCAGCCTTCCAGCCTCTCATTGCCCGAGG 130 cg03604011 AGGGAAGTTGGGAGTATTGTGCTCCCCTGAGAGCCGTTGCCACGGTTACG 131 cg12035880 CCAGCAGCAGCGGCAGCAAGGTTTACAATCCTAAACCATGCAGGCTTCCG 132 cg19601991 TAGCCATTTGGCCTCGGCATCACGGTGCGGGAGCGTGGGGCGCAGACTCG 133 cg26921969 CAAGCAAGACTCTCCACCCACAAACTGCATATTCTTTAAAGTCACTGTCG 134 cg04836987 CGCTTAAGGACCTTATAGCTAGCAAATGGCAGAGACAAGCTTGGGGCAGA 135 cg15658426 AGATGTTATCAGCTCAGCGAGACTTTCCATGATCTCCCTAAAATGAATCG 136 cg21777188 CGCCCAGAACTGGGACTTCCCAGGTTCTCATAGTGATTGTAGATATCAAT 137 cg05845376 GCTAGTGAGCCGCGCGCTGGGCCGGCAGCAGGGGGTTATTTTAGTGCGCG 138 cg07039560 GGCTGGAGGCGGCTGGGGGCCCGGCCAGTGCGCCAGGAGCCGCAGGAACG 139 cg25587069 GGCAGGCAGGCGGCAGGCAGAGCGCGCTCTCCGGGCAGTCTGAAGGACCG 140 cg13667243 CGCTCCGCCGGTGCTCGCTCTATTTGAAAACGCTGACTGTTGGTCCCCCA 141 cg12363682 ACAGAACCCCAGAAAAACTGTCCCACAACCCCCAGTGAATGACCAAATCG 142 cg07412232 CGGAAGCTGCACCCTAAATTCCCCGGGACCTGTGGCAGGCCTTCCTGGTG 143 cg15174951 AGCCACGAAGAAAACAGTTCAACTCAGACCAAAAGATAAAGCTGATCTCG 144 cg18892128 GCCTGGTCCCCGAAAGCGCTCAGTCAGAATCCGAGAGCACTGTGGCGGCG 145 cg14924781 CGCCTCTCACAGACTGCTTCCCCAGCAGGGTGCAGCTTCTTACAGACTGG 146 cg25955180 TGATTCAGTCCAGTGATTGGGTTTGTGGCTCCAGGCCTCGCCCACAGACG 147 cg27067781 CGTCTCGCCTTGCGAGCAAGCTCGGAATCCAGTTCCTCAGGAACCCCTCC 148 cg21091547 CAGCAAAGAAATGACTATAGTTGGAACACAGGACTTTTGCCTCCTGCCCG 149 cg26470115 CCCATCCATGACTTCTTATGTGTGCTGTCGGCAGCTGACCAGCGGCCACG 150 cg04344923 AGGCAGCAGCCCAGTGGCCTCTTGCTGTGGAGCGCGGGGGCAGCCTCCCG 151 cg06413272 CGGGTGTTGTGTCTCTGCCTATCTTCACACATGCACCCTTGTGAACTTGG 152 cg02817030 CGGACTCATTGCCATGCATGCCTAGACATTAATATGTGTTCATGGAGGGC 153 cg26403206 TGTGCTCAGATTCACAAAGTGAAAGTGCAAAACCCCGTGTTAGCTCATCG 154 cg15777014 CGGGCTATAGCGAGACCCCCCATAGTGAGATCTGCTGCGGGACCTAGAGT 155 cg01704252 CGGTGGCCTGTTGAGGCCCAACTCATGCCTCTGGCACCCTTTCAAGAGGC 156 cg15600935 GCAAGGTGAACCCACCTTGATCCCGGGGCTGCAGAGCCAGTGCGCCTGCG 157 cg25949550 CGGTTGGCAAGAGCTTGGCTAGAGAGACTGATGTAGATGGCAGCTTCTTA 158 cg11207515 CGAGAGGCATTTCAGCAGAATTTTGAAGGTGGAGTGAAGGAAGTCACTAT 159 cg09159795 AAACACTCTCCCGGCTCAGAAACGGTGCAGGTGCATTTACATGTCAATCG 160 cg07338119 CGCGGGCTCTATCTGCAGCATCCACCCCCTCAGCAGACGCTGGCATAAGC 161 cg09608412 GGGGCCGCAGTGTCCTTGGCTCAGGCCTCCTACGCAGGGAAGCAGGGTCG 162 cg14700531 CGGGAAAGTATCACTTGTGACTGTTTGCACTAGGTGCAAAAGACAGATAG 163 cg03900851 TCTGTAGAGAAGTCGACCGAGAAGTGAGATGCGGTGGCGACCTCGGAGCG 164 cg27272363 CGCTGCCCATGCCCAGACCAAAAATGGGGTGTCTTTTTAGTCTAAAAGGA 165 cg16821867 CGGCCAGACCCGTCACTCAGATCCCCGCACCAGGCCAGTGAAGAGGAGCC 166 cg15410835 CCAGCCCTTCTGGCACAGATGCTGTGCATGTGAGGTGCTTCTCAGAAGCG 167 cg00844308 AGGAGATGAGCTATGTCTTCCAACCTCTCCTTTTGGAATAAAACCTTTCG 168 cg13728106 CGACGACAAAGAAGATTTTGATGAAGTTCAAATTAGAGTCAGCGTGCTCA 169 cg27087885 GTAAAGTGACACCCATCCCACTCATCCCATTGCTGCCAATGACTTCAACG 170 cg20448001 CACCTTCCACTTTAAAGAACACCAAGGTCGGCTGGGTGTGGTGGCTCACG 171 cg15480287 CGAGCCCAGGAAATAAGTGCGGGTGACCGAATTCAATCCTGCATCCAACA 172 cg19414383 CGGCCCCACTTAGCCTGAAACGGGGCCCCATGCAGGGTAGAGGCCTGCCA 173 cg25536676 CGCACACCGAGTGGCACGACCTGCAGAGGTTACCGCCAGGTTTCCATCCT 174 cg22820188 TGGGGTGAGCCACTTTCATTTTCCCAGCGGGGCCAGGCAGTCTTTGCTCG 175 cg08410533 CGCAGGTCTGAGAGACATCACAGCATCACAGCGTTTAGTAGTCTTAAATG 176 cg12483947 CGACTACCACTGCTGGTGCAAGGACCAATCAGCGACCTATTGAAGTGCCT 177 cg23371436 CGGGATGGTGTGACAGGCAAGCCAAAGTTGTATCTGAAAACTCTGCAACA 178 cg24894584 GGTCCGCTGGGGCAGAGTGCGGAGTGAAGGGGTGCACTGGGCACTCAGCG 179 cg26266429 CTTTAAGATGTCTGACAGTGCCCTAAGGGCACGTGCTCCACAAACAAACG 180 cg10068417 CTCAAAGAAAATAAGCCAGGACCAGGTGACCCAGTGGGATCCGCGAAGCG 181 cg25572904 TGCGGCCACTGCAAAGCGATGTAAATGTCCCAGAGTGGGGCAGCTGGTCG 182 cg13543355 CGTGCCACCTGGCCAACCTCGAAAGGAGGCCCTGCTGGGTTCTTCATCTT 183 cg26481784 CGGCGGACCCAGCTGACCAAAGGAACTACAGAAGAGGACGAAGAAAGCGA 184 cg13898384 TCGCTTTCCAGCCTTTTGTTTCTGGATCCTCAAAACTCAGAACGTGGCCG 185 cg19459791 TGGAGCAAATATAGGTCTAGACACGTAGGGCCTGTGAGGTTTTCCTGACG 186 cg03172657 GCGTGTGCTCAGCGTGTGGTTACCGTGTGCACAGCACGAGCTTGAACTCG 187 cg18181703 CGTGGCCACTCTTCAGCATCTCTGTCGGAAGACCGTCAACGGCCACCTGG 188 cg07573872 CGGGAATGGTGACTCAGCCTTCCAGGAACCTGCGTGGCGTCTGTTTTTTT 189 cg09051966 CGGGCGCAGCCCCAGGATGCAACAGCTCTCATACAATTGGCCAGGAAGGC 190 cg25841091 CGGGTTTCCCAAGTGCTGGACGCGCAACAGGTTTCCATAAACCCAAAGYT 191 cg12516875 CGGCGAGTGAGTGAAAAATTACATTTTCACTTGGGTTCTGAACATTCAGT 192 cg10753966 CGGGGAAACTCTGAATTGGTGGTTGCTTAGAAAACACATAGAAGTGTGTA 193 cg23944298 TCAGGGYTGAAAACGCAACAGCCAAAATTAACTCAGTGGATGGCTCAACG 194 cg07553761 CGCCGGTGGCCGACGGCTTCTGAGGAATTATCTTTTACTTGGCGCCACAC 195 cg10202557 CGAGATTAAAGATTCAGAAAGTGAGTAAAGTTAACACTGAGCCCTTGGGC 196 cg05604079 AGTTGGTGAGCCAGGGAAGTCTTCCCTCGCCCTGAGGGTGCGGGGAAGCG 197 cg11444009 CGGGGCCAAAGAGGAGCGCAGGTTTAAAAAGCAAGTGTATTAGCAAGTTT 198 cg25149516 CGCCCACGCGGACCCCTCTCCGTGTGCTTGTCTGTACTTCCCTGATGGGG 199 cg18584747 CGGCCGGAGGGCCCTGAACCCTAGGCCAAGACTCATCCACAACCTAGGGG 200 cg20405584 CGGGAGATCCTCCCGCGGCCGGAGGGCCCTGAACCCTAGGCCAAGACTCA 201 cg11299854 CGTGAGCGGGATCCTGAGTCTGAGTTGCCGCTTAACCCGGCATTTTATAA 202 cg24708145 CGGAGGCAAGCAAGCTCCATGTGGTGGGGACAGCGCATTGCGACCTCCAC 203 cg25663874 CCATGCTATAAGTGAGGTAAATAAATATGCTGAGCACCATCGCACTTACG 204 cg25325512 CGCGGCATTCAGCAGAACTCATGATGAAGGAAACAGTTCCATGGCAGGGT 205 cg13001142 CGACAAAATCTGACCAGGCTAGATCTGAATCTGTTAATTTCTGAGACGGT 206 cg06649410 CGCAGAGGAGAATGTCAGATGCTCAGCTCGGTCCCCTCCGCCTGACGCTC 207 cg17501210 CGCCCGATTCAGACAGCTGGACTCAGAGGGATTCTGCTCCACAGAGAAAC 208 cg03473532 CGTATGTGTTTGAGATAGCAGTTGTTTACTATCACTTGAAAATTCTGAAT 209 cg20468415 TCATGAACTGGAGCCTGAAGTGGCTCTGAGGCCTCCCCTGGAAAGGAACG 210 cg11782409 CGGTGGATTCCTCTCGGGACACATCTGCCACCCGCTGGACTGGCAAAACA 211 cg07205627 CGACCATTTGTATGTGTATCTATGTCAGAAAGAATCTTTATTAAAATATT 212 cg07274490 TACCATGTAATATACCACTAAACACACACTATGTAATACACTACTAAACG 213 cg07929447 TTGTCTCAGATTACAGGAACCAAATGCATGTCTGGCCTTGTGCTGCTTCG 214 cg00695391 CGAGCCCGCCCAGCCTCTGAACCTCCGTGTCTTCATCTGTAAATTGAGGG 215 cg16305333 GTGCTCTCAGTCCAATTCACACTGAATGAATGAATCAGTCCCAGCCATCG 216 cg00970752 CGCTGAGGTCCCGTTTCTGTAGCTGAGTAGCCCGGTGACACTGCCCTCCG 217 cg04967775 AGGGCCCCCTGGGCCTGTACCAAGGTCTGCGACAGATGGGTGGGGAGCCG 218 cg06901711 CGCCGCAGTCCCAGTCGAGCCGCGACCCTTCCGGCTGCCCCCACCCCACC 219 cg01431830 TGTGCCTTGACGGGCTGTCCCAGGCGCCCCTGGAATTGATGCAGTGTCCG 220 cg10578779 CGCAGAAGAAAGCTGCTCAAGTGCTGGAGTGGCCAACCCCATGCCGTTCC 221 cg26359730 CGCACATCCTCAGCTAGCAGCATGGAGCTGAGTTCAGAGGAGGCACTTAG 222 cg10585941 CGGGAGGGAAAGGTGTTAGATGGTGGTTTTAACTCACCAGGGTTTGAATT 223 cg01939962 CGGCCTGATTCGAAGACTGGAATAGAGGATTTCTAGAATTCTGTGACTCC 224 cg06809285 CATGTCCCAGCTCAGCTGGTCCTGAGAAGCTGCCTGGTAGACAGTCACCG 225 cg11336382 TAGAACCAACTGGATACTGCACCCCTCAGTATTGGGGCGTCACAGCACCG 226 cg08410533 CGCAGGTCTGAGAGACATCACAGCATCACAGCGTTTAGTAGTCTTAAATG 227 cg04196119 CAAGTCCTTTCTGGTTCCCGTGGTTTTTCAGGTACTGCGGTTCTTATTCG 228 cg12329405 CTCCCTACGTGGGGGACGTGCAGGATGATGCGGGGTCGGGGGGGATTTCG 229 cg12379046 TCCGCAAAAAGCAGGATCCATTCACTTTAGAGAAGTGCCCTCAACAGGCG 230 cg03950121 TGTGGTCTCCCCAGACCACCAGGTGCTTCACACACGCACAGGGGCACTCG 231 cg03096347 TGCAGCCTCGTAAGCCTCATGGAAGAGCTTGGTTTGTATCCTTTGGAACG 232 cg22620356 CAGTGCTGAGCCGGATCCAGAAAATTGAACAGGTCCTGAAGGAGCAGCCG 233 cg17310258 CGTCCCCGAAATGAATAATGCAGGCAGGAGCCAAGATTTCTGCATTAGCG 234 cg18886347 CGGTGTGGAGGCTTTTTTTGTCAGAAGCTCTGGGGTTTGGCTCTTCTCTG 235 cg14592365 CCTCTTTGCTGATCTGGACTCAGCCTGGTATTCTTGTCACAAGGTCATCG 236 cg07631435 TTTTTAACCAGTTTGAACATTTTACTACTTCCTAACATCAGAAGCACCCG 237 cg03112631 GTGTGGCAAGGCTGAGCTAGTTCGTCTGCCAGGTACCAGAGCAAGCCTCG 238 cg01635346 CACTATGTTTGCTGCTTGCTATTCACTTATGGTACATACACAGTCAGTCG 239 cg25966649 GGGACGGTGGTAGTGCTTCTCAACATTGGCACTGGCTCCATTGTCATTCG 240 cg23510415 TGGGGAGCCAGCTGACTTAACAGGCCTGGATTACATTCTTGCTTCCAACG 241 cg18159533 TGCCATTTAATCTAACGCCACCTTCTACGGACCAGAGCAGGTGCTCTGCG 242 cg16982073 CGGTTAAATTCGTGAGGCTGTTTGCTGTGTACACAATGCTAAGCAGAAAA 243 cg25572904 TGCGGCCACTGCAAAGCGATGTAAATGTCCCAGAGTGGGGCAGCTGGTCG 244 cg02150207 TCTGGTCCTGGCCTGGGGGGGTCGTGGTCTCGCTGGTGCAGCCTCCTCCG 245 cg13649020 CGCCACCATCGAGTGTGAGCAGCCCCAGCCCGACCTCTACAAGTAAGCGG 246 cg06903325 CGCTGTGAAACCTTTGCCTTTGGGTGTCATGGTGGAAGCAAATCTTAGAA 247 cg05078817 CGCCAGGGCAAACATTCCTTGGGCGCCTCGGGCTTTTCCCTAGGGAAGCA 248 cg13898384 TCGCTTTCCAGCCTTTTGTTTCTGGATCCTCAAAACTCAGAACGTGGCCG 249 cg13634681 CGGAGAGACAGCCGCTGTGGTTAGGGGTGTGCATCCTTCTACCATGCCCA 250 cg01624571 CGTGGATCTAGCCACAGAAAGGCAACAGCGCTTCAAAGAGGCTCTCCGTA 251 cg14153919 CGCTTTTGCAAAACAGGCCGCACCTAGCCCAGATTCTGGAGCTGATTCAG 252 cg05575505 CGACTCTGAGTTCTGACAGGGAAATACAATGAGTTTTATGTGGACAGAGT 253 cg19987111 CTCTCTCCCAGACCTCAGGTAAAATTAATGTTCTAGGAACGTATGCCACG 254 cg05638439 TGGGGTGGCCGTCTGCTCCGTCTTTGGTGGTGAGAACCCGCACCGGTCCG 255 cg26470314 CGGTGTCTCCGCTCCTTGCCCGTGGCCAGGTGGTGGTGCTCCCAATGCCC 256 cg24429836 CGGGCTTTAAGGTGGCCCTTGCAGATCTTGCAGACATTTTCGTGGGCTCC 257 cg04120520 AGACTCCCTCTGAACACCTGTGGAACATGCTGACCTCCCAGTGTCTTTCG 258 cg09319676 CGCACATGTGAGGCCACCATTCATACCACACCCTGGGTGGGTCTTGAGTT 259 cg20433952 TCTGCTCATTAACACCCTGTTAATAGGAACCCCAGAAACCTGAGCTTCCG 260 cg02220125 CGGCCTTGCACACCATCTTAAGTGTGACGCCACACTGCCCTTCACAGCCT 261 cg18181703 CGTGGCCACTCTTCAGCATCTCTGTCGGAAGACCGTCAACGGCCACCTGG 262 cg10701033 CGCGCCTGGTGCAGCCCTCACCTCACCAGGTETTGATCATCTTAGTGGTT 263 cg07529654 CGCACTTAATATGATTATTTTTTGATGCCAGGTCCACCAAGTGAAGATTG 264 cg27145096 CGTGTAAAGGTCAGAGGTCCATTCACCAATGCAATGATCCTCACTGCCCC 265 cg07573872 CGGGAATGGTGACTCAGCCTTCCAGGAACCTGCGTGGCGTCTGTTTTTTT 266 cg14528319 TATTGCAGTATCCTAAAGCGTGCTGTATCTCTCTGTCCGGCTTTTGCACG 267 cg09920725 CGGTCTTGTAACAATTGGATGGATGCCTTTGAAGAGCCCCTGTCCCTATT 268 cg24000528 TCTCCTCCCCTGCACTGGCTCTGGGTGCTTATCTCTGCAGAAGCTTCTCG 269 cg13772414 CGGGGTCTAGGGAAGAGAGATTTCCTAACCTAGACTACAGGCATCAGGAG 270 cg08007899 TGATTCCTCCCCCTTTGAAAGAGCAGGCAGCATTTTTCCAAGGCAACCCG 271 cg15808795 CGCTGGTCTCGGGGGGGAACCCAACAGACTCACATTTCATGTGAAAGGAA 272 cg09845806 AAAGATGTCATTACACAAATACACAAATACTAGCATGCAGACACACAACG 273 cg03706056 GATGTATCTCCCACCCAGGAGTGTTTCTTGGCCAGGTGTGGTGGCTCACG 274 cg24110177 CGACAACTCTGTAGTGATTTAGAAGAGAATTGCAAGACATTAACACAGAG 275 cg10753966 CGGGGAAACTCTGAATTGGTGGTTGCTTAGAAAACACATAGAAGTGTGTA 276 cg00481951 GTTTCAGCACCTGGGTCAGCGCTTCCCAGGGTCAGCACCAGGGATAGACG 277 cg04748223 CGCAGCTGTCGTCTTTGTACCCGGCTTTGCGCACTCCATTGTGGACGCGA 278 cg22518157 CGGCTGTGGTTTCCTGTGCGTCCCAGGTGAGATCTTGCTCTTTACAGGGT 279 cg22714094 GCACCTTTGTGAGGTGTTTGTGGGGTTGGGGGAGCTTCAGGCGCTACTCG 280 cg10177080 CGGTACTTCCCTGATGGGGCCCACGCGGACCCCTCTCCAGTGTGCTTGTC 281 cg15845365 ATCTTCCCAGTACAGAGCTTAGCATACATTGAATAACTGGCATCTGCTCG 282 cg27628536 ACTCTGTCAGATATCCTCGATATTTGGGGATAGTAGTTCAAAAACAGCCG 283 cg24765579 AGCTGTTTCCCTCAGGCGGCCTGTTCCACGCCAGGATTAGTTTTGCTTCG 284 cg15988937 CGGTTTGCAGGTTCAAAGATGTCTAAAAGAGAATGGCCAGAATGTATATA 285 cg02081065 ATCCCTCATCTGGACTTTGAGTGATTGTGTTGCTGGCATGGACCCTTTCG 286 cg12612118 CAGAGCCCACTTAGTGCGCGCTAGCTGGGCAGGGATAGGGGTCCTATTCG 287 cg12298697 ACAGTATTTTTGTCACTCCAAGTATCCCTTATCTTGCCCTTTTATGGTCG 288 cg20660989 CTGAAAGGCGCATTGTCAGCCTGGGGAGGCGACGCGGCGCGAGGACAGCG 289 cg03546163 CGGAGGGCTTATTCTATGTAAATAGTTGAAAGGAACTGGATAAGACTGTA 290 cg25325512 CGCGGCATTCAGCAGAACTCATGATGAAGGAAACAGTTCCATGGCAGGGT 291 cg00684824 CGCCCAGCTGGAGGCTGAGATGTCAGGATTTGTCTTGCAAAGTTACTTCA 292 cg02713068 CGCAAAACTTAAGGCTATAGAAGATGGGGAGAGTATCTTTTGATGCAGAC 293 cg06649410 CGCAGAGGAGAATGTCAGATGCTCAGCTCGGTCCCCTCCGCCTGACGCTC 294 cg17501210 CGCCCGATTCAGACAGCTGGACTCAGAGGGATTCTGCTCCACAGAGAAAC 295 cg03807873 CGCTGCACTCAGCCACGTTACGCGGAGTTCATCTGGGGAAGGAGACAAAT 296 cg21899558 GAAGGGTCACACATGTCCCAGAATCCCCCAGGAGTGATGCAAATGGAGCG 297 cg07025011 AAACCCTAAACACTGAAGTCTCCTCTGGTCAGGTGACCCCTATCAGTCCG 298 cg16847637 GCCAGGAGTCCTCGGTCCTGGGAGGGTTTAAAAGCCAGGGGGCCGTCTCG 299 cg06563451 GACCCTGGGCTTGGGGGGATTTTTTTGGCCTGGAGGGCTCCTCTTCTCCG 300 cg02716826 GGCCAGACACCCAGGACCCAAATAACCTAATAATGGCTCATTTGCCTCCG 301 cg13947317 CGGCATTGGTCCTTTATTGAACATCCTCCCAAAGCTGGGGCTAGGGTTCA 302 cg22510139 CCAGATGTTTTCACTCGATTTTGCAAATCTGCTCCAGGTCTAAGACATCG 303 cg10116490 CGGGTCTTCACTCTGGTTGGCTGCCAGATCCTCCAGGGGCGGGGTCTCAC 304 cg27396830 TACAGAACCCCTGAAACATTCCTGCCAAATACAGTCGCACGAATTTGTCG 305 cg19414383 CGGCCCCACTTAGCCTGAAACGGGGCCCCATGCAGGGTAGAGGCCTGCCA 306 cg03345668 CGCACAGCAACTGGGGTGTCCTGACCAGTGAGCTAGAGTTCCACCTTACT 307 cg17719473 TTATAATCCAAAAATATTTATTAACCATCTGCTCTGTGACAAGCACTGCG 308 cg20569940 CTTTGGCTCGAGCTCCAGGTGCAGATTTACGTTCATTTTCTTTTGTTCCG 309 cg26156167 AAAAACTTTTCTGAGCCGGAGCCTCCAGCGCGCTGTGTATTCGTTTTACG 310 cg10578779 CGCAGAAGAAAGCTGCTCAAGTGCTGGAGTGGCCAACCCCATGCCGTTCC 311 cg15631106 CGCAGATTCCCCATTTGTGAGCAGAGTTAAGCTGAAGGAGCGTGGGCATG 312 cg22704788 CGGCCCCAGAAGGCAAGGCGATTGTTAGGAGGAATTAAACAGGCTAGTGC 313 cg20240347 CGCCCGGCGACCACTGGTGGTTCTATGCTGCTGGGGTGAAAAGTCTGAAG 314 cg17803430 AGGACACTGGCCAGCCTCCAGTTGGTTTCTGAAGCTAGCAGTCTTAGTCG 315 cg20800892 CGCCCTGCCCTGGGATTCTCAGACCTTGAAAGCCACAAGTCTTGAAAGTC 316 cg08410533 CGCAGGTCTGAGAGACATCACAGCATCACAGCGTTTAGTAGTCTTAAATG 317 cg12329405 CTCCCTACGTGGGGGACGTGCAGGATGATGCGGGGTCGGGGGGGATTTCG 318 cg14984434 CTGAATTCTGGTACCTGTTAATGACAGCTTCTGAGAGGTTTCTTTTATCG 319 cg07690734 TAACCTTGAACAAAGATCGTCGACGGGCGGCCCTCACTGGAAACCTTTCG 320 cg17310258 CGTCCCCGAAATGAATAATGCAGGCAGGAGCCAAGATTTCTGCATTAGCG 321 cg14955976 CGAGTTGTAAGTCAACCTGCTGGGTTGTGCTGAAGAGAAATTGGAGACTG 322 cg06975311 TTAGGGCCTGGAGATGGGCAGACCCAGGCTCACATCCTAGCTCTGACACG 323 cg16246188 CGGCTCTAAGCGGGGCAAGAGCTGCTTTTAATTTGAGACGTACACAGTTT 324 cg07631435 TTTTTAACCAGTTTGAACATTTTACTACTTCCTAACATCAGAAGCACCCG 325 cg12211040 ACACATACCCCACTAAAGAAGATTCACAAGGTTGGGTGCAGTGGTTCACG 326 cg11764442 ACTATGTACCTGGAAATAGCAGGGGCTCAGGGTTTGATCCTATGAGACCG 327 cg10535933 CGGCTACGAGCACAGAGCATCATCATCATCAGCCTGCCAGCCGGCCCCGC 328 cg01624571 CGTGGATCTAGCCACAGAAAGGCAACAGCGCTTCAAAGAGGCTCTCCGTA 329 cg24521756 CCTCTGGACAGTACTTTTGAGACACACTTATTTCTACCACGCACCTTGCG 330 cg07842062 CGAGAAGTTCAAGTTCAAGCTGCAGAACACCAGTGTGCAGAAGGAGCACT 331 cg08072101 CGCGCAGGAAAAGGGAAACAAACAGCAGCTTCGCAGCCTCCCTGGGCCTG 332 cg03955530 CTATGGTCAACTGAGTCAGGGCTGGATTTCTCACCCACCCTGGCCAATCG 333 cg07839457 CGCTCCTATTCACCGAACCCCTGGCTGGCTGACCCGGGAAAAACAGTCTA 334 cg04976151 TCCACCAGTTCTCTTATCAATGGACAGTACAAACAGTACTACCACGAACG 335 cg03400403 CGCGTGTTTCTCTGCGCTGTGGTCGTCATGGACCGGAAGCGTGTGCAGCG 336 cg04745805 TTTTCTTCTTTGGCTTTTCCATCACTGCTGGCTACAAGTGTTTGCAACCG 337 cg14545159 GGGGTTGCCGGCACCTTCTCTTTCTCCCCAGGTCCTTTCATCTGATTCCG 338 cg18181703 CGTGGCCACTCTTCAGCATCTCTGTCGGAAGACCGTCAACGGCCACCTGG 339 cg17100770 CGCACGGCCTCCAGCAGCAAGCTGATGCTGTCCTACAAGAAGCCAAAAGG 340 cg27145096 CGTGTAAAGGTCAGAGGTCCATTCACCAATGCAATGATCCTCACTGCCCC 341 cg13077366 GTCCTAAAGCACCAGGATAGATGACGAGCAGGGCCAAGTCCACACAAGCG 342 cg21656736 TTTAAGCTATCAATGTGGATGTGCTGGTGCTGAGACCCACCTCCAACACG 343 cg07573872 CGGGAATGGTGACTCAGCCTTCCAGGAACCTGCGTGGCGTCTGTTTTTTT 344 cg21736089 CTCCTCCGAGTAGCCGGCGCCGTGGATGATGCGCATCTGCTTGATGAACG 345 cg07543883 ACTGGACCCTCGGCTCTTCCTTGGACTTCTTGTGTGTTCTGTGAGCTTCG 346 cg10224107 TGGGGCTTTAAAAACCACAGCCCTTGGGCAGGAGGGACCTTCGATCCTCG 347 cg01909777 CGCTCCCTCTTAGGTCACCTGCCATTTCAGACAGGGAAGAGAACAGTGAA 348 cg03760035 AGCAGCTCCACCGGGGATGCGGCTCGTCAGGGGGTGTACGAGAACTTCCG 349 cg03706056 GATGTATCTCCCACCCAGGAGTGTTTCTTGGCCAGGTGTGGTGGCTCACG 350 cg13092901 CGCCACAGAGGGCTTCCCCAGGTCCAGGCCGGGCCAGCCTTGACCTCTAA 351 cg15849439 CGAACCTGATGTGGAGACAAAGTATCCTTCATGGACTTTGAATTTGATAT 352 cg12605080 AAGGGTAGCTGCTGCCACTGCTACATTCTACAGAAAGCTCAATGAGTCCG 353 cg01163330 CGGCTCCCTCACACCCACTGAGAGCATCATGTCCCTGGGCACGCACTCCC 354 cg10753966 CGGGGAAACTCTGAATTGGTGGTTGCTTAGAAAACACATAGAAGTGTGTA 355 cg19501902 CGCTGCTTCAACTGGAGTTAACCAAGTGAACACAGAGCTTTACAAAGAAC 356 cg13287247 CTCGGTCTCGTGTGTCTGTGTGTGCCAGTCCCTTGTGCTGACACAGGACG 357 cg22930808 TATGCAGTTGTAATTCTGACAGTATTGCCAAATTGTTCACAGTAAAGACG 358 cg08122652 CGGCAGAGAGGCCTGAGATAAGGTTTTTGCACATGGCTGGTAAGACTGGA 359 cg00959259 CGGTTGTCAGCACCATATTTCTAAAATGCAAATCTGGCAAGTCATTTTCT 360 cg20576510 CGCGGCTGTACCACATAGAGAAAGATATGCACTAGTTCAAAGAGAATGCC 361 cg10202557 CGAGATTAAAGATTCAGAAAGTGAGTAAAGTTAACACTGAGCCCTTGGGC 362 cg27134386 AGGCCCCAAGGTTCCTGACAGAGCTGCTGTTGGGTGTTGGTGTAAAGCCG 363 cg10584300 AAAAAGACTGCAGCTCCCATCTTAATTGCTCTCTCATGCTTGTGTGTGCG 364 cg03274876 GAATTCTGCTGGCGCTGCAGCTGCAGAATGGTCGGCGGTGGCGGGAAGCG 365 cg17287155 AGCTTCAGACACAGTCAACGTGCTGCCAACGTGGGGAGGGCCGGAGGACG 366 cg04764584 CGGAGATGAAATTAATGTGAACCACTGAGGCAAAGGCATGAGGCCCCCTC 367 cg15679283 CGGCTGCCCCCTCACCAGCACCCTCCGATTCCAGCGACAGCCTCTTGGCT 368 cg17533522 GTCCCTGGAGCCAGGCGTTAATCATTGACCTGATTAGCAAGAACGCTCCG 369 cg14868212 CGACATGACAAGATTTAAGGGAGGCACATCTCACAGACAAGCATGAAAAC 370 cg16274199 CTGGCCCGGCCGCCCCTCAGCTATTTGTTCACGTAATGCGATTGGAAACG 371 c213309828 TAAATGGACTTACTTTGAGGCCATGATCAACATTTAAATCTTGCTCTTCG 372 cg12612118 CAGAGCCCACTTAGTGCGCGCTAGCTGGGCAGGGATAGGGGTCCTATTCG 373 cg03415429 CGGACACTTCCGGTGGAAGGACTGAGCGGCGCTACACTTCAAGAATTCCG 374 cg15947697 TTATTGTTTTCCTGTCTTCTACAGCAGAATTGGATATTCCCAAACAATCG 375 cg11867651 CGGGGTCTGGGTTTTCTTGTCCCACTGGGAGTTTCAAGCCCCAGGTAGAA 376 cg17096289 TCAAGGGAGGGCGATATTCCAGTGCTGATCCCATTTTCCTCCCCTCCTCG 377 cg01517384 TCAGGAAAACTCATGCCATTCTCCATTCAACGGAGGGCGACATTCTAGCG 378 cg16928551 CGTCCAGCAGACAAAGACTATGGTTTAATGAAAATAGATGAACCAAGCAC 379 cg26987613 ACTGATGATTTCCAGGAACTACCAAAGCCACGGATCAGCTGAGTTACCCG 380 cg20660989 CTGAAAGGCGCATTGTCAGCCTGGGGAGGCGACGCGGCGCGAGGACAGCG 381 cg21288889 AACAACAAAAAACTTAAATTTCCCTTGACTTTTAAGTTCACTCTTGTTCG 382 cg06649410 CGCAGAGGAGAATGTCAGATGCTCAGCTCGGTCCCCTCCGCCTGACGCTC 383 cg17501210 CGCCCGATTCAGACAGCTGGACTCAGAGGGATTCTGCTCCACAGAGAAAC 384 cg02515033 TCCCACCCCTTCCATTCTGTTGAGCTGAGACGCGTGTTTTGGTCGTTTCG 385 cg00907204 CCTGATTTACACTTGGCTCCACCCATAAAGTTCCCACTGATTATCACACG 386 cg16847637 GCCAGGAGTCCTCGGTCCTGGGAGGGTTTAAAAGCCAGGGGGCCGTCTCG 387 cg03473532 CGTATGTGTTTGAGATAGCAGTTGTTTACTATCACTTGAAAATTCTGAAT 388 cg07205627 CGACCATTTGTATGTGTATCTATGTCAGAAAGAATCTTTATTAAAATATT 389 cg14508358 CGGTCACGTGATACCCCCCAGTGTACCTTTCCTGCAGCGTGTTACGTGGC 390 cg04661709 CTGGGGCTGAGACCCCATTTCTGGGAGGCTCTTCCCAGTGCTGGAGACCG 391 cg13428009 TCTTTGAGCCCCAGCTTCCTCATGTGTAAAACAGGATAATAGCAGTGTCG 392 cg21789941 CTGCTGGGGAGCAGTGGACGCCCTCCAGGGTCTAGGGAGCAGATAAGACG 393 cg22866430 GCACCCCCCCATCCACTTCATGTTCAGAAAACTCAAAGAGTCAGAAAACG 394 cg18234973 AGCTCTGGAATCCAACTGCCTGAGTGGCAAACCTATCTGTAAGACTTACG 395 cg26110733 GGGCCTTTAGTGCTGGCTCTTGAGTACCCAGACCCCAGTTAAGAAATACG 396 cg10636246 CGACACCCTCAAGGGAGGAGTGCAGGCACTCAAAGATTTGAGTCACAGGC 397 cg10578779 CGCAGAAGAAAGCTGCTCAAGTGCTGGAGTGGCCAACCCCATGCCGTTCC 398 cg17591574 TGGCCTTAGGTTCTAGTGGCTCTGGTGTGGGCCTGTGCCCAGCCTTTACG 399 cg13371627 TTTATAAACCTGCTATTCCTTTCCTTCGGATCCCTGGTTTTCCATCTCCG 400 cg12458003 CTGGGCTTGACAGCTCAGAGCAGTGTCCCCAGCTGTGGCCCTAGAAGGCG 401 cg13180152 TTTGCCTCTTTTTCAGTCTTGTAATCCCAAACAAAATAGAAGAGCCATCG 402 cg21459583 AGAAGCACGTGGCACCTGTCATAGGCCCCTCTGGCTGCTACAAGTTCCCG 403 cg18278247 CGGCACCTATCCCAGCAAGATCCCGGGAACAAAATCCTGGAAGACCCTAT 404 cg11295183 CGCGTGAGTGTCCTAAGGAGTCCAAGCACAACAGATGGCTGAGTCACACT 405 cg18388639 CGAGCCTGTTGTCAACATTAGCCCCGGGTTTCCCAGCACCAACTCCAGCG 406 cg06752157 CGGAGCGTGAGCCTAGATTCTTTGGAAGGCTGTACAGTTTAAAATCTAAT 407 cg18148156 CGGCAGGTTGGAAATCCAGTTTGTGGCTGATGCAAGCAAACCATGCTGCA 408 cg19935065 CGGTTGTGAGGTGCTCACGTGTTTTGGAGATAGCAAAAGTCTCAAATAAT 409 cg10549018 TGTCTCCTCATCTCCTGGATCTTTGCCCAGCAAAACCTCCAAAGAGACCG 410 cg07927953 CGCACAGCCAGCAGGCGCCTGGGCTCCACTCTCTACCTGGAATCACCAGG 411 cg01057893 CGGCAGTCTTCTCCACTGAGGGACTGGGCTGGGAAGTCCTGCGTTTCAGT 412 cg04431596 CGGCCCCACCCGGTTCTTGTGTCAAGACAAAAAGAAAACCCAGGTGGCCT 413 cg06806711 CGCTGATAGACATCAGGTGACAGGAAATCAGTAGCTTCTGCTACCTTGGG 414 cg13620770 CGGCAAGCATCATCGCCAGGCCCCAGGCCTCCTGTGGGACGCCAGTCACC 415 cg03764506 CACTTCCTGACTAAGGAGCTGCAGCGATACATCGAAGGGCTCAAGAAGCG 416 cg07582047 TCCAGGAAGCAGCAGAGGTCGCGCAGCTCACGGTTCTCTGCCTGCAGACG 417 cg16649728 CCAGCTTGAAGCCCGGTGCCAGATGCATAAGCCACTTGCCCGAGAGCACG 418 cg06854487 CGTAGCCAGTGCGAGGTTGGCTCTACTTTGGGTTTATGGGTTGGGCATAA 419 cg25371036 CGGGCAGCTCTACTGCTTGGAGAAGAGAAAGCTAGGTAACTGTACAGTTG 420 cg20941739 CACAGCCAGAGCACTTACAGTGAGCCGAGCCACAGTCCAGCAAGCTGCCG 421 cg05021075 CGACACTGTGATAATCATGAAGTTTTGTGTGAGAAAGATAAGGGAGAAGA 422 cg01975858 ATAGCTATTCATCCTTAGACCCTGTCCAAACAGAGCAACAGTCATATACG 423 cg20923047 TATTTTTGAAGGAGCTGGTCAGCATTTGCCTAGACTCTCGGGACAACACG 424 cg19783150 CCCTCTAGTGAGACCCATTAGTGTATAAGCTCTGCCTACACCGAGTTTCG 425 cg01641177 CGGCCATGCCCTGGCCCCAGAAGTCGTTGGTGAAGATGCCCCAGCAGAGC 426 cg07677157 CGGTGATCCATAGCAAAAGAGCCTATGAGTCAGGTGATGATCCATTCATC 427 cg21579274 ACTACTTTGGGCCTCGGTTTCCCTGCTCCTTGTAGATCAGAGAAGGGACG 428 cg01687878 CGGCCATGTCTGGTCACTGCGTATGAATGACATCTGTCTCTGCAACGCTT 429 cg18159533 TGCCATTTAATCTAACGCCACCTTCTACGGACCAGAGCAGGTGCTCTGCG 430 cg04323814 CGCAGTGACAGGAATAATGCCACTGTCCTGCTGAGTGGGAAAGGAGAGGG 431 cg04836038 AGAGGTCTCAGGAAAGTAGCCTTTATTTATGTGGCACCGATCGGAACCCG 432 cg16188984 CGCACAGCACGGAAAGGGAAGGCAGCAGCCACATGGACCTGGGCCCACGG 433 cg11605750 CGGCAACCCCGGCCAGACAGGGGGTATCCAGAGGGCCCAGGACACCCACC 434 cg18607491 AAGAACGACTCTCCGCAATGCGCATGACAGAAAAGCGGCTGCTTCTACCG 435 cg14870958 TCGAACGCTGGACCCTTGAGACATGTGACGGGACCTGAGGGCAGCCACCG 436 cg19491388 CGGAGTTTCTGTTACCTGCAGGGAATGCTCTCTTAACTAATCCAGGGGTG 437 cg02135821 CGGAGGCTGGGGAGATGACATCAGAAAACCTGGTCCAAACTGCTCCAAAA 438 cg09279803 CTTTACTCAATGCCAAGATCCACATTAACACAATTCCACTGAATCCTCCG 439 cg26481784 CGGCGGACCCAGCTGACCAAAGGAACTACAGAAGAGGACGAAGAAAGCGA 440 cg19459791 TGGAGCAAATATAGGTCTAGACACGTAGGGCCTGTGAGGTTTTCCTGACG 441 cg16730765 CTCTTGATGACCTCGTTGTCTGTGCTGGCACCATGGGTCTCATCCTGACG 442 cg19987111 CTCTCTCCCAGACCTCAGGTAAAATTAATGTTCTAGGAACGTATGCCACG 443 cg02880119 AAGTGTCAACGAGATGACTTGCGCTACAGCGGCACACTGGTGAGGAACCG 444 cg22616007 CGCATCTGCTTCCCATGAGTCTCTTGGGGAAGACAAAGCTCACATTCTTC 445 cg06946797 CTCAGGGATCTGTGAGCAGAGAGGCCTGGCTGCAAAGGGTCTGGAGGACG 446 cg26837962 TGTGACAGCCCTGGGCTGAAAGACTCCCGGTGGCCGTAGTGTAATAGCCG 447 cg08659340 CAACTGCTGGTCTGTTTTCTGCCCCTGTAGTTTTGTATGAATTCTTTACG 448 cg07984286 CGCAATGTAAAATTATGTCCAGTTGTAGATGTGTGTGTTGGGGTTTAACT 449 cg00417823 CGGGCTGGAATTGAGCTCAGCTAAACAAAGTCCTTCAGCTTTGAAGGGAG 450 cg05180206 CGGACAAGATAATTATGATATAACAAAAGCTTGTGTTATTTAAGGTGGGG 451 cg09899215 CGTCCCTGGAGCCCACGAAAATGTCTGCAAGATCTGCAAGGGCCACCTTA 452 cg04379563 CGTCCCGACTTCACGCGAGGGTTTATACCTGAACAGACACAGCAGGGAAA 453 cg09834822 CGCCCTCTCTGTGTAGCTGGAAGAGCCTGACCACTCACGTCTCTCCAGGC 454 cg18951537 AACAGCCATGATTCCTAAATATTAAACATTTTCCTCATCTGACAGGATCG 455 cg23842572 ACAGAGACAGAGCCCAAGAATAGAGGCACACGGGGAAGTAGACAACATCG 456 cg25613227 CGAGGGTAGTTGGTCAGAGTTGTGCGAGCTGGTTGCAGGGAAGGCTACCG 457 cg07660627 CAAGCTCTGTGCAAAATAATCTGTAGTGCTTGCTATAACGAGCTATAACG 458 cg22156456 CGGGGATAGGCAGGTAAGTAGGATCAAGGAGTGCAAACAGCCAAGTGTGA 459 cg18181703 CGTGGCCACTCTTCAGCATCTCTGTCGGAAGACCGTCAACGGCCACCTGG 460 cg25876186 TCGGGGAAGCTCAGGGTCTTGGGGTCTCAGGGCGGGGAGGCAGGACGACG 461 cg23691771 CGGGCAGATCCCAAGGTTTCCTGATATTCCCTACTGCTGCAGTCTCTGCT 462 cg00870265 GTCTTCATTGTTACTCAGCGGGGACACCCTCCTGGCGCTCCGGGGGCTCG 463 cg03739265 CGCTGGAGACCAAAATCTTGGGCTATGACCACCCTGGGCAACATAGTGAG 464 cg03636183 TGGTGGTGGGGCTGCCGGCCAATGGGCTGGCGCTGTGGGTGCTGGCCACG 465 cg02291424 CGCGGCTTGACTAGTAAAGTGCTCAACTATTCTCGACCACCATTGGGTAT 466 cg26853093 ATGTGGCGGGGGCTTTGGACCCTGGCGGCCCAAGCGGCACGTGGGCCTCG 467 cg03110633 CGGCCGCCGCCCCCACCCGACCCAACCGCCCGCACAAAAGGCCGGAGCCG 468 cg07127225 CGGGCTCGGTCCAGACTGACAACAATGCCCTCCTCGTACCTGAAGAACTT 469 cg06022832 ACGAAGGACAGTGATCCCTAACAGTTCAGCAGTCCCTACTGTGGCAACCG 470 cg21566642 CGCCTCCTGAGCCTGACTCTGCTCTTCCCGCAGCTTTGGCCCCCCGGGGC 471 cg01940273 GCACGGGGCCTGGCACGTAGGGGGCCTTCATTGCATCTCTCTTCCCTTCG 472 cg08110610 CGGGTGGGGACTAAACAAAAAGGTCAAGGAGAAATGTCAACCTCAAGATG 473 cg00684178 CGCTGGGCTCTAGCTCTGTATCTGTGCTCCTGGCAGGCCAGCACCCCATC 474 cg24580066 TCAAACTGCCGCGGGCGTGCGGGTTGACGCCGAGTGTGTGTGTCGGGGCG 475 cg04588969 TTCCAGTTCGTGCCATCAAGGAGGGCGACCTGAGCACCAAGTACGATGCG 476 cg21727276 TGAGATCTCTGTCTCTATCCTATCCTGTCCCTGGCCTTCTGAGGCAAGCG 477 cg09051966 CGGGCGCAGCCCCAGGATGCAACAGCTCTCATACAATTGGCCAGGAAGGC 478 cg18345924 TGCATACTGATTTCTGAACATTAAATCTGCATCCAATCGTGTTTCATCCG 479 cg18638931 AAAACACCAACAAAAAACGACTCCTAACATCACTCTGGGTGGTGAAAGCG 480 cg27380813 CGATCCGGAATTCAGGATGAGCCATGAAGTTATGGATGGAGGTTCGAGAT 481 cg03827386 CGTTGTTGTTGTTTTAGTTACGTTTAGCTTTGTCAGCAACACTTTGGCCC 482 cg15849439 CGAACCTGATGTGGAGACAAAGTATCCTTCATGGACTTTGAATTTGATAT 483 cg06349851 CGGCCAGAGTCTTATCCCCTGGCTCTGGCTGATGAGGAACACGTAGAATG 484 cg25598319 CGCGGGGGGTCCCCCTGGCACCTCCAAGCCCATCCCATTGACGAAGTAAC 485 cg25845889 CGAGAGGGCAGCTTTAAGACATTTTAGAATTGTTTCTTACCATTGTCTTT 486 cg15228492 CAAGTCCAGTTTTCCACCTAAATATCACTTGAGGCCAACATTCTCTTTCG 487 cg03900798 CACCTGACAGACAAAGCCCTGGGTCCAAAATGGCTCATCGGGGTCAGGCG 488 cg20576510 CGCGGCTGTACCACATAGAGAAAGATATGCACTAGTTCAAAGAGAATGCC 489 cg12988996 TGTGCCTCCCTGGCAGTCAAAGTTCTAAGGGGGACTCCTGAAGCCAGGCG 490 cg11569478 CGGACTCGTTTTATTGACTGACAGTCAGTGCTAGCAGTGACTTCAGCTGC 491 cg01603456 GTCATAGAAGGCTGTGTTGCCCCCAGCAAGAGCAAAGCCACCGCTCCACG 492 cg18584747 CGGCCGGAGGGCCCTGAACCCTAGGCCAAGACTCATCCACAACCTAGGGG 493 cg17543884 GAAAACCCAGCAGAACATAACCGTGCAGGTCTCCGACGTCAATGACAACG 494 cg22090064 CGCAAGAGAAGAAAAAAGGTAAGAGTTTAGTTCTCAAAAAGACAAAGAGT 495 cg11923914 CGAAGGAAACCTTCGCTGGCTCCCTAGTGCCCTGGAATAACACTGAAACC 496 cg18394552 TTTAGGCAGGTGGCCAATTTTGGTCCAGCTGGTGGCAAGGGCAGGTGTCG 497 cg24801535 CGGCAGGTTTTGGGACTCATTCCTGCAAGCCCAAGCCAGAACTTGTTCTG 498 cg12325588 CGCCATTCCCCAGCACCCGCAGCGGGCACTGTGGCTGCACGCCGCCCTCG 499 cg23367341 CTGTCAGTAGATCTCAGCGGGAGAGCAGTGCTGGTATGTGAATGTCCTCG 500 cg04987004 CAGTCCCAGCTTTCACTGCCAGGGTCCCAGTCAGATTCCAGGAATTTGCG 501 cg24859433 CGCCCCTGGAGAGGCCTCTTGGGGAATGAACTGTCCCTCCCAACCTTGGA 502 cg10940833 CGGCGGCAGGAACTATCAGTAGACAGCTGCTGCTTCCATGAAACGGAAAA 503 cg11539695 CGGGCCCTGCAGTCCTTAATAAAAAATTGCAGATTTCATTCTAGGTGTCA 504 cg26987613 ACTGATGATTTCCAGGAACTACCAAAGCCACGGATCAGCTGAGTTACCCG 505 cg17598713 AGATCCTAGGTGAGGGCAGGTTGACTGGCAGGGCTGGTTTTTCGGGATCG 506 cg26433444 AAGGCCTGGCTGGCTGCGAGCTCAGGAGGCCGCCTGAGGACTGCACACCG 507 cg25325512 CGCGGCATTCAGCAGAACTCATGATGAAGGAAACAGTTCCATGGCAGGGT 508 cg02272457 CGGCCCCACAAATTACGTGTGTGCAGGATTAAGGGAAACCAGCAAGGGGT 509 cg10169261 CGCGCCTTTTATTGACGCAAACCTCCCGTTCTCGCGGGAACTTGGGAACG 510 cg03098356 CGCCATCTTCACACCCCGCTCCGTCGCGTACGCACCTGGGTCACGTGATA 511 cg06649410 CGCAGAGGAGAATGTCAGATGCTCAGCTCGGTCCCCTCCGCCTGACGCTC 512 cg15822010 CGGCCCTGCACACGTGTCCACTGTGACGCGTCTGTCATGGTCCCCTCGGC 513 cg15089567 CGGCTGGGGACCGTAGCAAGAGCAAGAGGGCTCCTCAGCAGTCCCTGTGG 514 cg00332048 CGCCAGTATGGAAACACAGTGTGTAAAGCAAGCTTCGAGAGAGGAAAGAG 515 cg01391867 CGCCTACAACTCTCTCGGTCAAACAACCGAAAACATCCCGTGCCGGAAGC 516 cg07113802 GGTGCGCCCTGGGCAGCTGGGTGGCCTCAAGCCCTACTGGCGAGACTCCG 517 cg03473532 CGTATGTGTTTGAGATAGCAGTTGTTTACTATCACTTGAAAATTCTGAAT 518 cg15573979 GGATGCACTGCGAGCCTGCCAGGAATTCCAGGCCTCGCAGTGTTCTGGCG 519 cg08202754 CGCTTCCACATTTGGACGCCCCCACCCAGCGGCCTCCGTGGCACACACCG 520 cg14260002 GGAGGACTTGGGGTGTGGCAGGGGAGCAGCCAAGCCGGCAGATGACTACG 521 cg06563451 GACCCTGGGCTTGGGGGGATTTTTTTGGCCTGGAGGGCTCCTCTTCTCCG 522 cg03460603 ACACAGAAATGGGGGTTCTCACACTACAGATTAAGGAGTGTCCCAGGCCG 523 cg25448355 CGGGACAAGAAACAACATGTGAGCAGCACAGCCATTCAGTTGGGATGCTG 524 cg24493971 GGCGGGCCTTGCCTCCAGCAGGGGTGTGAAGAGCCCAGGCTTATTTTCCG 525 cg02716826 GGCCAGACACCCAGGACCCAAATAACCTAATAATGGCTCATTTGCCTCCG 526 cg14355192 TCCTGCAGGCCTCACCATAGGGCACCTCTTTGGCCCAATTCAGGGCTCCG 527 cg13649864 CTCTGAAGCCTGCACCTCTCCATTAAACACAGAACAGCTGCCAGAGGACG 528 cg23549061 CGGTGTGAGTGCTCACTCAATGCAATGTGTTGGTGATGGTAACTGTCCTT 529 cg13947317 CGGCATTGGTCCTTTATTGAACATCCTCCCAAAGCTGGGGCTAGGGTTCA 530 cg21272576 CGCTGCTGCCTGGGACTCAGTGATTATCCCATTTTGCAGACCAGAAAGAG 531 cg03522107 AGCCGGACAAGTGAGACCAGGCGGCCCCTTGGGTGGGCAGGGTCTGTCCG 532 cg03259703 CGGCTCAGCTTGCTCACCCACCCCAGAAGGAGCGATTTGCATTTATGAAG 533 cg05461666 CGGGCAAGGAAAGGATACAGGTATCTGCCTTTCGCATGGCACTGAGCAAA 534 cg15860924 CGGGACAAGCTTGACAAGGTGGGTGTCTTCCTGGACTATGACCAAGGCTT 535 cg10327168 CGGTAGAGAATATTTTGACTCCTTGAGTAATGGGAGTTCTTTTGTGGTCT 536 cg24393673 CGCGCTGCAGTCACGCCTCCCGCTGCCAGCCCGGCACCGGGATCTTAATC 537 cg23923856 GGAGGAGGATCCCCGAGAGGGGCGCAAGTTGCGATGTCCGGAAGCCGACG 538 cg14476101 CGCCAGCAGATACAAAGGCAGACAAAGTGAGCGAGGCAGTTTCCAGGACC 539 cg20569940 CTTTGGCTCGAGCTCCAGGTGCAGATTTACGTTCATTTTCTTTTGTTCCG 540 cg10578779 CGCAGAAGAAAGCTGCTCAAGTGCTGGAGTGGCCAACCCCATGCCGTTCC 541 cg05996213 CGGGTGGAAGGAGAATGAGGAGGCAACCAAAATAAGATCAAAAAGTCAGG 542 cg03424844 CGATGAGCAAAATCACACATCCTCATGTCCATCTGGTGCTCTGTATTGTG 543 cg19570545 GCGGAGCCAGCGCTCTTGGGGGCAGGGCTGCGAGTCCCCTGTGTGGGACG 544 cg20248954 CGTGACCTGTCTGTACCCTAGCGCGGGGCTTCTGTACGCTGCTAGGAAGC 545 cg08138571 GAAGCAGTTAAGACAACAATACCCAGGACTAGCCAGATCCCGAATTACCG 546 cg23814988 CGTCGGGCTCACAGAAGGCTCAGGTTTCCTCCAGTATAGTTCAGGGGATG 547 cg20801751 CGCCTCATACAAGAACACAGAACAGGGAGCTTGTCTCCTTGGGGATTAGA 548 cg11333189 CGCTGCGGCAAACCTAGGAGGGACCCAAAAGCAGAAAATCCTAGAAAAGA 549 cg08055490 CGGAAACTGATCTAGTCAGGGAGGTCAGAGAATATCTCCCTGAGGAAAAG 550 cg02613108 CGCACTGACTGGTGAGCGCTTTGCACCAATTAGTAGCAGGTGCTACAGCC 551 cg12329405 CTCCCTACGTGGGGGACGTGCAGGATGATGCGGGGTCGGGGGGGATTTCG 552 cg20245361 CGGGGTGCATTAGCTGTGACTTTTGCTGCATCCAGTCATGCACATGTGCC 553 cg07793207 GCTTTCTGCTTTCACCGGGAGGCCCTTTAATTGTTACCAAAAATAGGACG 554 cg07733920 AGGTACTGGATGCACGGCAGGCCCTGCCACTGCTTCTCAGAACCCTCTCG 555 cg02415341 AATTCTTTCACAGTTCTGGAGTCAAGAAGTCCAAAATGAAGGTGTTCACG 556 cg12893697 CGGCCCTACACGGGCACCGGGCCTCAGTCCTGCCAGGCCCACGCCATCCT 557 cg18886347 CGGTGTGGAGGCTTTTTTTGTCAGAAGCTCTGGGGTTTGGCTCTTCTCTG 558 cg19083871 GAAGCTGGCGACGAACCCGTGTCTCAAGAATTTCTACGGCTTGTTTCCCG 559 cg13875901 CGGCTGCAGGCCCCAGAGTCCACGAGGGTGGGTACAAATCCCCTGGGCAC 560 cg02970861 TGATTGGCCCAAAGCATCGTGAATATTCATGACAGCAGCCACAAAGGCCG 561 cg26967875 TTCTCGGGTCTTCGTGAGCATTAAGTCAATGAACCCAGGGCACCAAAGCG 562 cg00574958 AGGCCAGCGAACATCCAGCTGTCAGTTGGTCTGGGGACTATCAGCATTCG 563 cg22355889 GAAGATGGTGAACCTGAGATGACCTGTCAATGGGATTCCCAGACAGTCCG 564 cg05839709 TGGATAAAGGGGTTTCTAATACCCAGCTCACATGGCTGTGGTGACAAACG 565 cg19061798 GTGAACACCTGCCGAAGCCTTTGCCTTGGGAGAGGCTCCAGCAAAATCCG 566 cg11932091 CGGGCACTGACTGGGGACCCAGGTGAGTCTAGCAAATGACAAGGCCCAAC 567 cg26527903 CGGCAAGGTTTATAACGAGAATAAATGAAATAAATACAAAATCCAGAATA 568 cg24420742 CGGGGACTTGATATGGAAAGAATTAATGTACTGGCTTTTTTGTATAGATG 569 cg10948795 CGGGTAGGGTTAGGCTCAGTTCAGGCAGAACCTGCCATTCGCTCATTCAC 570 cg17117459 ATTCATCTCTAGTCTCTGTAAAATGCCAGCCCTCTTCTGGGTTAACAGCG 571 cg18331061 CGCACCGTCTCACCTGAGTTTTCTGCCACTGGATCATGTGGGTCTGGTTG 572 cg21879240 GAGTGTTCCCCATGCTGACTTCTGCCAGGCTGCAGGGAATAAAGCTAACG 573 cg20656525 CGGAAAGGTTCATTGGCTAATTTGCCACAGTTGGTTTTCAATATGTCCAT 574 cg14543641 CTGTGCAGACGCCATTTGCCCCCAGCTTAATTGAGCATATCTCTCCCTCG 575 cg04634427 CGGTGGGAACGGGAAGCTTCCCCAATCAGACTGCATCCTGAGGGACAGGA 576 cg01624571 CGTGGATCTAGCCACAGAAAGGCAACAGCGCTTCAAAGAGGCTCTCCGTA 577 cg05575505 CGACTCTGAGTTCTGACAGGGAAATACAATGAGTTTTATGTGGACAGAGT 578 cg06051159 CGGCCTTTGTGGATGCCAACCTTTGTCACAGCCAGGAGGGGGCCTCCCTC 579 cg00994306 CGGCCTCAGGCCAAGCATGAGAACAACGTCAGACAAATCCCCATTGATTG 580 cg02295856 CGCCGACCCACAGGGCACAATGGTCTACAAAGTTGGAAATGAAGGTCTGC 581 cg12991522 GCACGGGCAGCTGAGCTGGGGGTACAGGGCACCCCCGTGTCCCAGGGTCG 582 cg00036119 CAGGAAACTCCTACCTCATCACGGCAGCTGCATGTGTGGGTAAGCCCACG 583 cg05995267 CGGCAGTTCTTAGTGAGGGCAATTCATTCAATCCAGAGCCAGAAACAGGA 584 cg06722193 CGGCCACCTCAGACTTCTGTATAGGAGGAGGCCTGACTGGGAGATCCCTG 585 cg05782444 TGTCCCAGGATGACATCAAGGGCATTCAGGAGCTCTATGGTAAACCTCCG 586 cg26943759 TATGGACAGTCTTTTGCCTGGCGTGGGGGCCAAGTGGACAATACCACACG 587 cg05737638 CTCCCTCCACGGGACCCAGCCTGCAGAAGGGTCCTGCAGGAAGTGGCACG 588 cg21649005 GAGTTTCTACTCTGCCTACAGGTCTCGGGTGAATTCACACAAACTCTTCG 589 cg06419850 CAAGATCCAGGGACCTAGAGGCCTCGGCGATGACACTGCGCTGAACGACG 590 cg19761014 CGCCAATGGTTCCAGCAAAGCCCCAGGACTGCTCTGCTCCTGGTCTTGTA 591 cg05174890 TCAGAGCTGTTTTTTTTCGTGGGTAACTTCCAGTTGCTCAAGGGTCCACG 592 cg10431713 CGGCTCCACACTTCCCCAGCGTGGCCTGCATTTCTATTGGGCAGCTGGCG 593 cg02483931 TATGAGAGGCCAGAATATTGCTTCCGTTCCCTTTTCATCTCTGCACACCG 594 cg07477034 CGCCCTGATAGCACCGCAGAGGTTCTGATCTAATTGATCTGGGATGCGGC 595 cg15815084 ATATACAGTGGCCATGAAGCAGGCACCACTTAGAAACCTCTGTATTAACG 596 cg12054453 CGGCAGAAGCTTCACCACAAAAGCGAAATGGGCACACCACAGGTAAGACT 597 cg03568017 CGGCTAGAGACTCACTGACTCATGCGTGTGTGGTAGGAATCTTCCAGGAA 598 cg02633924 CGGGACGGGGACTTCCTGGAAGATTCCTACCACACACGCATGAGTCAGTG 599 cg18181703 CGTGGCCACTCTTCAGCATCTCTGTCGGAAGACCGTCAACGGCCACCTGG 600 cg27637521 GGAAACTTGCTGTGGGTGACCATGGCGCACGGAGCCAGCGTGGATCTGCG 601 cg05420896 GGGTGGTACAAGGGGCGCCCCTTCGCTGTTTTAAAATCTGATGCTTCACG 602 cg19283806 GATTTCTCCTTGAACAATCCCCGCAAAGATAGCAGCCAAAAAAGGATGCG 603 cg27569863 GAAAGCACTAAATTATCCTGCAAACTCCCGGCTGATGCCTAAGGAATTCG 604 cg13611456 CGGAAGCACAGTTTGCAACAGTTTGGGGTTGTCTTATTTTCACTGCTGGC 605 cg10677351 CGCACGGATGGGACCTGCTTCTCACTCCTGCACAGCTGAACCTCCAGGAC 606 cg15011409 CCAGGAGTGCGCTGCTCTCGGGAAGGCATCCCATGGCCTGAGCAGCAGCG 607 cg17676631 GCTGGGTCCCCGTGTAGCACTTAGCTAAAGATGATCAGCAGCTGAGAACG 608 cg11594160 ACAGTATACAGTCGGTGCTCAGGATGCATTTGTTTTCCTGTCCCCCAGCG 609 cg26950531 TGCCGGCCACGTGGTTGCTGGGCAGGTGTGGGGTCAGGCTGTGGATCCCG 610 cg22538557 CGGCTCAGCCAACCCCCTAGATAATAGCGGTTTCTCAAACTGTGGTCCCG 611 cg09920725 CGGTCTTGTAACAATTGGATGGATGCCTTTGAAGAGCCCCTGTCCCTATT 612 cg10354495 CCTATTCTTCCAGAACCTTCCTGGAGTATTCCGTTTTCTCTAGGACAACG 613 cg04659537 CGGGGGAATTCATTGATAAGGAGAGCAAGCATCCCAAGAACATCCCCATC 614 cg14088844 CGGACACAGCACCAGGTCCAGATTGGCAAGAAAGGACCCTGGGCTTGCTC 615 cg07258300 ATCTTCAAGTCTCACTTTGGTCCTCAGTTTGTAGTATCTATTGCAGACCG 616 cg26191447 CGGTACCAGTAACTGCCAGGAAAAGGAAGGGAATATGTCCAGAAAAAGCA 617 cg02831419 TGGTTATTCCCCAGGAGCCGCGCGAAGCATGAGCTAATTTTCAGTGAGCG 618 cg20550050 CGCACATGAGGCACAGAGAAAGGACAAAACGCAGCCACACCTGAGCTGCG 619 cg06266189 GTGCCTCCCTTCTGCACTGTGGGTGTGTGGAGCTGTCTTTCTTGGAGACG 620 cg12791136 TGAGCAGGAGCGAGACAGAGCACCACGGGGCACGTGAGGCCAGGGCACCG 621 cg23278885 TCCCACAGTGAACCCCTCTAGGTGCAATTACAGGATGATTTTGTGTCTCG 622 cg08074820 GCCCCAGGACCCCGGGATGTGCAGCCTAGGGGTGGGGTTGAGGTTCTGCG 623 cg11086127 ACCTCTTGTGGTATTCCTCTATCAGGCAATCTGGGTGGGCAGAGACTTCG 624 cg00412842 AGTCTTAGCTTAAGGAAATGTGCCACCTGCGTGCAGGGATGAGGTCATCG 625 cg27294156 CCATGCTGCGGATCCTGTGCCTGGCACTCTGCAGCCTGCTGACTGGCACG 626 cg22163406 TTCATCGCAATGGGCTGTATAGCTTGGCCCGTCTCATTGTAACAAATTCG 627 cg08334432 TGGGCTAGCCCAGCGCCACCTTCCTGGAGACTGTCTGGTGGAAGACACCG 628 cg06133392 CGCTGCTGAATGCACAGCACTGTGAATGCACTTCCATCACTGAATATACA 629 cg23573129 CGGGGCTGAGTACTTGGCTGACCACCCTAGGGCCACACCAGCCCAGGGGA 630 cg20585830 CGCACAATACGTGGAAGGCCATGGAAGGCATCTTCATCAAGCCTAGTGTG 631 cg19253577 AAGCACTTGATGTTTGCCCTCACACTGTTCCTCTCCCATTCCAGTTGTCG 632 cg09219182 GGCCTTACACCAGTGCAGAGGGCAGGCTTGAGGCTGATCTGGTACACACG 633 cg01708387 TAGAATAGTGACTGGCACATAATAGGTGTTCAATAAATGTCAACCCACCG 634 cg12918464 CGCCTCAGCTGGTGCTCCAAGACCTGCTGGCCAGACTGGTGAGGCCAGGT 635 cg14508777 CGGGGACAGGGCTCCTAATGTGTTTGTCAAGTAAATAATGAGTAATAATC 636 cg05993589 CGGTGAGGCCAGTGCACTCAAGTTTGGGAACTACCAATATAGAGTAAAAG 637 cg23431721 ACATACGTCACATTGGCAGTGTTTGTAGGATATCGGGCTGTAGGGATCCG 638 cg07105440 GCTAGCTGCCGGGCATTGGGAGGGCTTGACGGGCCAGAGGTGACTGAGCG 639 cg03929796 CGGCACCTCGCCTCGACCTTGCCATTTTATACTCAATTGGGGCGTAGGGT 640 cg23511118 GCAGGTCATTACTATGGGTAACTGGGGCTCCATCTCTCTGAAATACCACG 641 cg13155421 CCCTGGCTGGTGTTTCATGTCCACCAGCTCTGCGTTCAGCACAGCACTCG 642 cg03840504 CGTCTGTCCCAAAGGGCGACACAATCCTGAGGCAGCTGTTCGCAAACTCG 643 cg19389001 CGGCGCACCTCTCCAGTGGGACCCGCAGGACGCCGTCTCTCAGCCCCACG 644 cg15709989 TAAGGCCCTTCAGAAATAGGATCGACAGGGCTGGAGTGGAGGGGCCCCCG 645 cg13296371 CTAGCATGGTGCCTAACAGCTCCTACCAAATAGTAGATGCTCAGTAAACG 646 cg21761813 CGGTCAGTCTGCCTACAGAGTGCTTGCCAGGCAATGAAGGAAGAGCATGC 647 cg00398048 CGTGCGAACATGAACTAATAATCCCTTTGGGTGTCTGGAATGCCTGCCCA 648 cg25149516 CGCCCACGCGGACCCCTCTCCGTGTGCTTGTCTGTACTTCCCTGATGGGG 649 cg10177080 CGGTACTTCCCTGATGGGGCCCACGCGGACCCCTCTCCAGTGTGCTTGTC 650 cg09578605 CAACACTGTGAAACCCTGTCTCTACTAAAATACAAAATATTAGTCAGTCG 651 cg01817885 CGGTAGGGTTTGGAGGTATTATTCAGTGTAGAGGAAACAGTATGCATAAA 652 cg12980795 CGCTGGCAAGATGAATCACAATAACAACTCTGAAGGCAAGAGGATTGGTG 653 cg01256539 CGGCAGAATTGTGCTTAATAGTTGGTGAGGTTTCTATGGGCGATGTGGAA 654 cg19076659 CTTCCTTCAGGGAGTCGTATATATGAGAGGGCTTTAGCTTGCTCTCCTCG 655 cg05182912 CGGGACACGTACTATCTGCCTCAGCCTAGTGCTAAGCACTTTACACATTT 656 cg00543335 CGGGGCTCATGGCTAAAGTTAAGGTACTGATCAAAGTTTTGGATGTAAAT 657 cg21584251 CGCTCCAGGCAGCGCTCCACTTCCGCGGCTTCGCCCTTGACAGCGCCCTG 658 cg00558975 GGAGGGCATCTTATTTTACCAGGTAAACAAGGGTCACTAAGAGGCATGCG 659 cg01187498 TTCACACTTAGGGCTCAGGACGCCCCTGTAGGCAGGGTGCTGGTGGGACG 660 cg22983827 CGCAAAAGCAAGATTCGCTCCAGGAGTTTCATGCCACAGGATGGTCGACG 661 cg02782857 GCTCATCACCTGGGGGCAGCTTTTGTACCTGCACCCTGTATGAGGCTCCG 662 cg17286012 GGTCTCTGGCATCTGTCCCGTCTCCAGCACAAACAACATCTGGGCAATCG 663 cg02066343 GCTGAGGGTGCCAGGTTGTCCTCCAAGGCCATCAGAAAGAAGGCACCACG 664 cg25325512 CGCGGCATTCAGCAGAACTCATGATGAAGGAAACAGTTCCATGGCAGGGT 665 cg08373528 CAGCCTCATGAACTCCATGGGTGTCGTCACGCTCCTCATTTGGCTCTTCG 666 cg15290848 CGTTCAGTGCCACTCAGGCCTGGCTGCGAAGATACAGATGGTATATGTGA 667 cg08877853 GGCAGTGTTGACTGCGTTCCATACCGGGACATCCAACACAACATTTGTCG 668 cg01883195 TGATTCTGGACCTCAGCTCTTCAACAAAGCTGAGGTCCAGAATCAAACCG 669 cg00936722 AGCTTTCTGGGTCTCCTTAATGAGCCTGAGGCCTGCTGCCTGCAAGTGCG 670 cg00554421 CCTGAGGCTCTGGGAGCAGATGGGAGCACAGGCTCTGTTTTGGCCCTGCG 671 cg11287647 TGCAATGATAAGTTTGCTGAACTGCATCTCTGCCCAATGCCACTTCCTCG 672 cg13917964 CCCATGCAGGAGAAACCCGTTTGGTGTTTACTACTAAGTCATGATCTCCG 673 cg00277397 CGCTGTGTGTGTTGAGACACACGCAACATTCGTGCACATAGAACTCATGA 674 cg20352798 CGGTGCCCCACAGGCCACTGTGCAGGATCTCCACTCGGCCCTGACACAGG 675 cg10119082 CGGGTCCCCAGTCCTCAAGGTCCCAGCACAATGCCCCAGGAGGAAGACTC 676 cg17981101 CGCCCCAAGTCATGAGGGAAGCTTCCAGACTGACTCAGGCTCAGGCTGGC 677 cg03494277 CGGTGACTGCAGGCTCCTGGATCCCATGGAGCAGAAACCTGAACTTGTCT 678 cg09315887 TGGCTACCGTGCTGTGGGGTGCCTCGAAAGTGTGAATCACCAAATATTCG 679 cg04599158 TGTTCAATGTAATAAACAACTGGCTCTTTGCTGCTTCAGATCTGTGCTCG 680 cg02627240 TTATCAAAGACTTTATATTTTCTGTAAAATACTACACAGTAAATCTTCCG 681 cg01026009 CTACCCTACTGGAAAATAGTGTTAATTATTATTCAGTTCTATTTCCATCG 682 cg03276920 TCTTCTTTCCACAGGTTCTCAGGCAAGAGCCACCTGCTATTGCCGAACCG 683 cg13398470 CGTGGCTATGCTGTGGTCTCCCTGAGCCTACTCTAGCTGGAAAGGTAATT 684 cg07205627 CGACCATTTGTATGTGTATCTATGTCAGAAAGAATCTTTATTAAAATATT 685 cg05065162 CGACACTGCACATCACCGAGACCAGGCACAGTGCCGGTTATGCCTAAGCA 686 cg12396523 CTCCCGAGTTCGCCCAACTTACTACTCTGCATGCTGGTGCCTAGTCTGCG 687 cg12831076 CAACGGAACGAAGCTGCATCTAAGTCTTATTCTAGAATTCTTTGTGACCG 688 cg22902266 CGGACAATTGGGGCCAAACGAGGAAGGACACAGACCCAAAAGCCAGACCC 689 cg04661709 CTGGGGCTGAGACCCCATTTCTGGGAGGCTCTTCCCAGTGCTGGAGACCG 690 cg01355108 AACAATTATGGGCTTAAAGAATTGATCATTACAGCCCCTGGGATTTAGCG 691 cg26581729 CGCCATAAGTGCCCCCAGAACTTCAGCGCCCACCATGGCGCACAAGGCCG 692 cg18476633 CGGTGAGTCATGCTTCAGGTTTGCCAGGGCCTGTGTCCCGCAAAAAGGCG 693 cg26341773 CGCTGGGAGGCACCACTTGACATGACCTGGGACAGGTGGCTCAGTCTCTC 694 cg25034591 CTCTGCATCCTCCCCAAGCAAAACCTCACCAAGGTGGTGAGATCCTGACG 695 cg03102848 CGGGGGCGCCGCCTAGAAACACACAATGGCCCCTCCTGGCCAACCCCCCG 696 cg18154457 CGCGTCATAAAGGACAAGCAGACCCAACTGAACCGCGGCTTTGCLTTCAT 697 cg11924796 CAACCTCAGGTTCCTTTTAATGGAGTAGTCACTTATTCATTCAACATACG 698 cg08871010 ACAGTCATACTGTCCTGAGTAAGGACAGTCAACAAAGGATCATTTCTCCG 699 cg06780601 CGCGTAACACTGTGCCTGGCTTTGCAAATATATTTTACAGATAGTGTAGT 700 cg27395754 CTGACCAAGGCATCACAATAAATACAGTTAGCAGCACACAGAAGCCTGCG 701 cg02587153 CGACATTAAAAGACCAATGGAGAATGTATATTAGCAATCTGTAAGGCAGT 702 cg15192905 CGCCGCCATCTCAGGTCTCTTGGCTTTGCCAGGGCCCACCGGAGAAAACT 703 cg08352336 GTCAGTTTTCTCCGGTGGGCCCTGGCAAAGCCAAGAGACCTGAGATGGCG 704 cg01997410 GAGAGGCGCAGGCGAGCTGGAGGGGAGCGGCGCGCGGCGGCCAGTGGGCG 705 cg16730484 GAGAGTTCAAAGTGAGCTTACTTCCAGTCTCACGTACAGATGATTCTACG 706 cg22686892 CGGGATGGAAGTTAGCGAATTTGGGCAACTTGCCTCTACTGCCCAGTCGA 707 cg14073590 CGTGGGCGAGTTGGTGGCAAAATCTGAACTGGATACTCACGGAGGGCCCG 708 cg07989867 CTTTGGTGCAGAACCACCGCATCCACTTTCTCAGGCCCAGGGGAGGGGCG 709 cg21080294 AAGCAGGCATGGTTGATTTCCCGCTAGCGGGAACGATCTGGCCTCAGACG 710 cg14680768 CGGCAGAGAGTTGCCACTGCAACGCCTGGCTCCCAGCCCTCCCACCCAGC 711 cg10142252 CGCCATCTCAGCCCTGGTGAGCCCCAAATCTGAGAGGGAGGGAAGGTCCT 712 cg18717447 CGCCCCCAGCTGCCAGTCCCCAGCAGCTCAGTCCTGCAGTGAGAGTCTTG 713 cg03960747 CGGAGGCCAGGCCACGCAGAACCTCACAGACCAAAGCTGCTTCGCAGTCG 714 cg16832801 CCTGCGGGACTACCAGAAGGTGCTAGTGCAGCTGGAGAGCCTGGAGACCG 715 cg11781622 CGGCCGGGACTCTCCATGTCCTATGGACTCTCGCTGAAGCCACTCGGCCC 716 cg13947317 CGGCATTGGTCCTTTATTGAACATCCTCCCAAAGCTGGGGCTAGGGTTCA 717 cg24145109 GCGGTGTGCACTGTGACCCGCCTTCCCGGGAAAGTCAGAGCACAGAAGCG 718 cg19694465 CGCCCCTGCCTGCTCGCTGCACTCCAGGTCACAGTTGGCACAGCGCACCG 719 cg15211499 GAGGAGTGGGTGGGAGACACGCGTGTCCATGCCGCGAGGGTGTGTCGACG 720 cg10511249 CGCCCTCGGAGCCCCTGCAGTCCCCTCACCGGGCGCTCCCAAACAGCCCG 721 cg16383389 GCCGAATTTCTGAGGTATCCGGGCTCTGGTGGACTGAGGGGGGCGACGCG 722 cg04607246 CGGGGAGGGGGTAGTTGTACTCTGCTTGTACAGTCCTTGAGCCCAGTTTA 723 cg08926056 TCCCACTTTTGTGAACCTTGAGTTTTTGAAACTTTCACTGAAAAAAACCG 724 cg14652203 CGCAGGAGGAGTTGGGAGGACAAAAGGAGAAAATGAACTAGTTGGGTTAC 725 cg20248954 CGTGACCTGTCTGTACCCTAGCGCGGGGCTTCTGTACGCTGCTAGGAAGC 726 cg23814988 CGTCGGGCTCACAGAAGGCTCAGGTTTCCTCCAGTATAGTTCAGGGGATG 727 cg14626196 TCTTTTAACCCAGCTAACACTTCTGTTAATAGTGTCTTCAGCCAGATGCG 728 cg16704703 GGGGCAGGACATCTCCCAGACAGCTGGGGACACCCTTTCCAGCGGGAGCG 729 cg19328485 CGGTGGGGACACAGAACCAAACCATATCGTTTGGTATTCCAGAATTTCTG 730 cg04254609 TCTTTGCAACGTCCCTCAAGCACTTAAGGAGAGGCGGCAGGGCCCAAGCG 731 cg02974659 AGCAGGACAGGCACCCGCTGGCCGGGAGGTCATTGACTGGCAGAACGACG 732 cg19795482 GGAGGAGGACTTGAATACACGCGTCGCTGACTCAAAGATGGTCCCCTCCG 733 cg17133967 CCCGGAGCAGTGGGTTCCTCTGCCGGGCCAGGAGGGTGATGGAGGGACCG 734 cg17639894 CGCGCCGGCTGACGTCACCCTGTGTCGACCAGCCCCCTGAGCCCGGAAAG 735 cg13657511 GAAGCACAAAGGCGACGTGACTCCTAGAATCTGTCCAGAACCCATGGTCG 736 cg26411299 GTGACAGCTGAGCTGAGACCTCAAGACACAGTCACCTGGGCCACCTTCCG 737 cg05425050 CTTATGCAAATCACTTAGGTACATGCAAAAGTATCCCTTCTCCCGGAGCG 738 cg11058730 CGCGCCGCATCTCCGGGAACAACAGTCTCCCTCCCGAGCATCACAGCCAG 739 cg05590616 CGCTCCTCCTAGCTAGCGGCCGCCGCCCGCCGCCGCCTGCGCCTCCAGCT 740 cg07268726 CGGCCCCAGCCTGCCCAGGGCCCAGCTGCTGGAGACCCGCAGCTCGTCCC 741 cg02780017 CGGAGGGCTCAGGTTACACCCCATAGGAGGGTTCAGGTGTCCCCACATCT 742 cg05710301 CTCCAGCTGGCGCACTACAAGGTAAGAACTGACCGCCTAGGGTTTTGTCG 743 cg00574958 AGGCCAGCGAACATCCAGCTGTCAGTTGGTCTGGGGACTATCAGCATTCG 744 cg02580722 CGCGCAGTTAAAAAAGAACTCTTALTTTGGTTGGTTGGAAAAGAGCTGAT 745 cg11507178 GTCAGAGTTCGGGTGTCTGTGGGTCTCTGAGCCTCTGCTGGCAGCACCCG 746 cg08832227 CGCAGCTCAAGACCCTGGCGCAGTTCCCCAACACGCTGCTGGGCAACCCT 747 cg22047262 CGGCTGGCAGGGGCCCTGTGCCCCCAGGTACCCCGTACCCCTGCCAAAGT 748 cg11043909 CGCAAGACTAGGCAACCTCCAGCCAGTCCCTGGGTCGGGCGGATCCTCCC 749 cg06705834 AACATGAGACAACTGAAGTGCCCTGGTTTATGTGCCCTGCTCCTCCTCCG 750 cg17177219 ATTTCACAGATAAGGAAACTGAGGCCCAAGATGGCACAGTGGCCTCGACG 751 cg24805559 GAGACATGGCGACGGCGAAGGTGGCAGCCTGGAGCCTTGGGCTGAGGTCG 752 cg10323552 CGCCCAGCCCAGATTTTGGCAGTGCCGAAGCTCCACACTTTCCTAAGCAG 753 cg13649020 CGCCACCATCGAGTGTGAGCAGCCCCAGCCCGACCTCTACAAGTAAGCGG 754 cg01936220 ATTGTCTCCCAATTTCTTCGTCTGTAAAATGGGGCTGGCAGCAGTGCCCG 755 cg09225388 CAGCCACTACTCCAGACTTTGTTCCTACAACTGACGATTGCCAGAATCCG 756 cg05241355 CGGATCCGTTTGAAACCTGTCCGGTCCCTGCTTTCTATGCCAGGCAAAAA 757 cg21112391 CGTGGGGTAGGGACTTTCATCAGGACATAGAGTGGCAGTCATGTGCTTCG 758 cg12155356 AGTGTCCGAGTAAGCCTAATGACACACATTCACATAACTAAGAAGACACG 759 cg08140104 AGTCCACGTGTCAGAGGTGTTTGAACCAGAGCAACCCTATCTTAAATACG 760 cg01823956 ACTTTCTCCTTCTTGTTTTTATTCGGCATGGCTGGAGCGGGCCGCCGCCG 761 cg10439456 CGCAGAGAGATGTACCAACACTGTGCTCCTGAGTGACTTATAAAGAGCCA 762 cg21791657 TTTAAGTTAAAATTGCCCAGACGTGGTATTCTGAATCTGGGTGATTGGCG 763 cg19510604 CGCTCCTGGTTGTCACATTCTGGCTCTTTTATGGGGTGGGAAATCTTTCA 764 cg07120314 CGCTGCCAGGAGGAGCCGAGGCGAGTGGGCGTGGCTCTGGCGAGTGCCCG 765 cg10530344 CGCCAGCCCCAAGCAAGCCAGGCAGTGTCCTGGTGTGGCAGCTCACAGGC 766 cg21658515 TCATCCAGGCTGCAGTGCAATGGAGCGATCTTGGCTTACCATAACTTCCG 767 cg16708495 GTGGTGGAGATGCCTGACATGGCTTGGGGACCTGCTGCCGGGGGAGCTCG 768 cg03997502 GTGCAGGGGGAGCTGGCGCAGCTCAAGGCCTGGGTGAGGAAGCTGCAGCG 769 cg08405844 CGGGGCTCTCCCAGATGCCCACGCTGCCTGCTCCCACTGGAGACGGGGAC 770 cg05144928 AATTGGAATGTGGCAGGGAAACGCTGGGAGACCCCTCCCCATCCTTCTCG 771 cg09351263 CGGCAACATCACTCCAGAGAGCCCAGCAGCTTCCTGGGAGAGGCAGGCTG 772 cg22510362 GAGGGTCAGGTCTGTGCTGGGGGTGAGGTCCATGCTGGGGGTCAGGTCCG 773 cg03072681 GTCTGGTCAAGAGAGATCCAGACAAAATCAACCTTCTGCGTCAACAGCCG 774 cg06959205 CGCAGGGCACTCGGAGCAGGTCTCTGGCTGCCTGGCACAGGAAGGGCCTC 775 cg00892703 CGCTGATACTGACATTTACAGATGAAACCGTCTGAGACCCGCTTCCCAGC 776 cg06912355 AGGGTGTCGGCAGCAAGATGGCTCCGGGGGTTTAGACACTGCTGGCTTCG 777 cg13606988 CGCACCCCAGCCAGAGAGGGAATACTGCACCTCCCAGAATGTGGCCTGGG 778 cg16569347 AAGTACCCCTGCCGCTATTGTGAGAAAGTGTTTGCTCTGGCGGAGTACCG 779 cg12165215 TCAGGAGAAATTTCTAACTGTAACCAAAGGGAGCAGGCAGGACCCAGACG 780 cg08667899 CGCGGCTCAACCAAGCCTAGAAGTGGGGGCGGGGAATCTTCCGCCCTCCT 781 cg01130991 CGGTATTGTCATGTTGCATCAAGGTTCCTCATTGCAGACAGAAATCTGCC 782 cg12054453 CGGCAGAAGCTTCACCACAAAAGCGAAATGGGCACACCACAGGTAAGACT 783 cg18181703 CGTGGCCACTCTTCAGCATCTCTGTCGGAAGACCGTCAACGGCCACCTGG 784 cg10508317 AGCTGAAGGTCTTGAGGCGCAGGCTGGTGTCCAGGGGGCGGCTCATCCCG 785 cg27637521 GGAAACTTGCTGTGGGTGACCATGGCGCACGGAGCCAGCGTGGATCTGCG 786 cg04243822 CGCACCCAGCCCTTTACAAGCAGCACAAACGTATTTGGTTGTCTGGGGCC 787 cg12486944 CAGGAGGCATGTGGGGGCCTCTGCCAGGCACTGCACAGGAGGGCGCGTCG 788 cg24391240 GCCTCGCGGGATTAGGGGCCGGTAGAGGTGGGCGGGTCGGGCGGAAGTCG 789 cg19617213 CGCCGAGGCCCGCTGCCCGCCCCCGCCCGCCCCCGCCCGCCCGCCTCACC 790 cg12846567 CGCCCGGCTGGCTAGACCATTGCTAATGGCTCAAGAGYTCAGTTCTAGCA 791 cg03358468 CGCCTGCAATTAAAGGCCAGATTGGAGGCATCTGATCCCCAGCCCTGATT 792 cg18569704 TGTTGGACCTCTAAACGGTAATTACGGGCGACAACGCAGATCCAGGATCG 793 cg15001547 GAGCAGCAGCAGCATGTACCTGGCCATGCTGGACGGAGATGCTTTGAACG 794 cg02991799 GCAGCCGCAGAGACCCTTGGGAAGTGTGGTCTTCGGGTGCCGGCGGTTCG 795 cg25450266 CGGAAGGGATGTTGTTTTGCTTCCGCAGACACCTGACCTCCAACTTCCTA 796 cg22030032 CGGGAATGGTGCTGTCTGCCCAGCCCCACCCGGCLTTGTAATGAACACTT 797 cg09915601 TGCCTGCAAACTAAGGCAGCATTATTCAGACCTGGGCTACCCTGGTCCCG 798 cg05971102 CGGTCTGGYTAGAAAATATCCAGCCCCACAGCAAGGGGAAAGAGTAACAT 799 cg07520608 CGGATTTCTGAAAATGTGGTTATTCCAGAGAATTCCTTCACGAGATTTAA 800 cg01412762 CGGGTGCTTCCAACCAGTCTGCTGCTCCTGCCCCACCCGTGCACGACAGC 801 cg09920725 CGGTCTTGTAACAATTGGATGGATGCCTTTGAAGAGCCCCTGTCCCTATT 802 cg12977946 GTGGGCGTAGGACCGTCTGAGCCATGTGTGGGATATAATCTCCTGGTGCG 803 cg20150163 CGCCGGAGGCCGCCACAACGCAGGCGCATTCAGCTAAGGACCACTCCCTC 804 cg04946715 CTTGTGGCCATCTCTGTAAAGTTTCTGGGGAGGTGGGCACGGGGTAAACG 805 cg08645207 GCATGGCAAGAAGCACCCCCAACACCCAGTTCTCCTGGAGACACTGATCG 806 cg14716323 CAATACAAATTAATAACAACCAGCCAATTCCATTGTCCTTGTATGCATCG 807 cg01956624 CGCGGGCAGGTGCTGCTGGTGGGCGAGCTGTGGGAGCGCGAACAGAGCCG 808 cg00497251 CGGGACAGCATGTCACTCCGGGAAGCCTCCCGTTCAGGTGTCTGGTCCCT 809 cg27114661 AGCCAGGAAGCAGTTGGCAAAGGGGCGGGGTGACGCGGTGACGCAATCCG 810 cg03443360 CGGAACATGTTTGGGTATAGGGAGCCATCAGACTGCAGTGCACACCTGAC 811 cg17758363 CGGCCCCTGGATCCTAATTCAGACCAGCCTGCCTCTGGCAATAGGAAAGT 812 cg14928764 TCTAGTGTCTCTAGGGCCACCTCACTTTCAGCAGTCTGTCCCAGGTGTCG 813 cg11850311 TACATCTGCTCCCCTGGGCTTTCTCAATTCCTAAATAATGTTGCTGTTCG 814 cg14234805 GGAATGGGAGGAACGCAATGCGTCCCGCGCGGCGATCTGGGATAGCTGCG 815 cg20747455 CGGGAGCTGTCAATCACGAACACCAGATCCAGGGGCCCAGTGTGACACCT 816 cg12112556 CGCTCTCCTAGAGAGGGCTTCTGATTTGGAGCTGTTGTTTTGGTCTGAGA 817 cg17262492 CGGGCCCCTGGGGCCTCAGAGGGACCCCGGCTGCCACTGACATATGAAGA 818 cg07871532 AGTGGCTGCAGCATGGGTCAAGGTCAGAGAATCCGGGAGCCCCACAGCCG 819 cg08548559 AAGCGTACGCTCTGTGCCAGTCCTTAGGTAAAATGTTTTACTTTGTGCCG 820 cg27115863 CGCCAGCCCGCGGAGCCCCAGTGACTCAGAGCCGTGGCTCCCCTGGCCCG 821 cg22650271 CGGCTCAGGACCAGCAGGCATGCCTCCTTGGTCTCATGTTGACCTTAGAG 822 cg19778944 CGCGACTCACCCACGGTGGCCGGGCAGAGCCTGCCTCGAACCCGGCTTCT 823 cg17025841 GGAGGAGGCTTTCTGCTGGCTAGTACTTTTCCAACTCTGACAGTGGCACG 824 cg06841024 GAAGCACTTGGACAGGCCATCGGGCCAGAAGCACCTCGCAAGGGTGTCCG 825 cg27326062 TACTTGCTCAGGGCTTTCTTCCCGCGTGTGCTTGGCGCTGCTCACAAACG 826 cg10747042 AGATGCCTAATTCAGCCGGGAGCCGCGGGCCGGAGCTGGCTGAAGGTCCG 827 cg18098839 CATAAACCAGATGTTTCTTAAAATAGCCCAGTTAAATCCACCCTTCCTCG 828 cg13457961 CGGGCGGGGAAAGCGAGGTTTAACTAACTCCGCAGCTATATTCTTCCGCC 829 cg10407935 CGCCACCCCACACTCCCTGCCTGGGGAAGTCGGACGCCGCCCCACACCCC 830 cg01096266 CAGAGAGCAAATCCTTCTTCCTGCCCTAGGCTGCAGTGCTCCTAACCTCG 831 cg05613002 TAAACCGAGTTAATCATCGGTCCATTTGAATAATCAGTCACGTGGTGGCG 832 cg05890887 CAAGCAGCCAAGCTGCTCCACGATTGGCTGCTTTAGCCTTACGTGCGTCG 833 cg06440348 GCAAACCGTGAATGTGGGGCAGTGTGGGGCCAAGCGGTGCGCTTGGTACG 834 cg21833476 TTTCTCGAGGCAGGGGGCACGGTAGCACAGGGAGCTTCTCTTTGTGGGCG 835 cg26036479 CGACAGCCACCTTATGTGGAAAATACTCGCGCCGGCCGTCATTCATAATC 836 cg25629712 CGGCTCAGAGCACAGGCTCAGAAATCAGACTGCCCACGTTTGAAAGCTGG 837 cg12716346 GGCGGCGCTGGGCATTCTCTGCCATCCTTAGGGGTCCTCCACAGCATGCG 838 cg02514143 CGCACGAACCCTTCATCCTGCTCCAAACAGAATGAGAATGTCATGTCGCA 839 cg15016740 CGCTGCCCAAGTCTCAGATATTCGATGCCTCACATTGCCAATATGGAGTC 840 cg01256539 CGGCAGAATTGTGCTTAATAGTTGGTGAGGTTTCTATGGGCGATGTGGAA 841 cg08260658 CATGGATTTAATCAGTGTACTTTTTACCTTCAGGCTGTAAATTAGAAACG 842 cg25024993 CGAGATTTTTAACCTGAAACTGAAGAATCTGGTACTGTAAGTGTAAAGAA 843 cg22157525 CGCCGAGGGCTCCAGGCCTCGCTTCCAGCCATATAGGTAGTAGGCCGAGA 844 cg21227060 CGCCTGCTCTGTGCCCCACTACGCTTCATGCAGGGGTCCATGTGCAGCTC 845 cg17255947 CAGTAACTCCGGCCGAATTTGTTCGGGTGTGAGGTTTAAGGCCCTAATCG 846 cg26372792 GTGACTGGCCAAAAAAAACCGCAATTTTGGGGTCTAATTCGATTGTGACG 847 cg26867866 GGGCGGTCAGGCGGGGTCAAGGCCAGGCAGCGGGGCGCGTCTGCGTTGCG 848 cg09144073 CTATTGGTTGAGTTAACCAGCATTCAAAAGCAGTCAGCGAACCAACTACG 849 cg11074353 CGGACAGGTTTTAAACAACCTACCAAGAGCCAGCGTGGGTGTCCACACCC 850 cg10000096 CGGGGTGAACGTCTGGGTCCTCGGAAATCCTCAGTCTCTGGAACTGAATT 851 cg17501210 CGCCCGATTCAGACAGCTGGACTCAGAGGGATTCTGCTCCACAGAGAAAC 852 cg26960221 GGGAGGCCTTGGTTGGAGTGTCTGAGGCCTCAGCTGGGCCCCTGCCTCCG 853 cg24172278 CGCAGCAGCCAGCTCATGGGCCCCGCATCCAGGTGTGTCCTGCCCTCTTC 854 cg01753176 CGCTGGGCACTGCCCTGGGGGCTGAGCAGCTGTCTCCATTGGTCATTCAG 855 cg21843627 CGGAACTCGCTGGAAGCTGTGATAGTCCCAGCTATGGTTTATCACAGGGA 856 cg19080320 GAGCTGCAGCTGGGGACTGGTGCGCCATGCAATTGACATAGCGCTGCTCG 857 cg20915897 TACTTCAGGGTAAAGAAGTTCAGGTCTGCCATCTCCAGCTCTGTCTGGCG 858 cg26116499 CGGCGGCTTCCCCAGGTGCTGCTCCGCCCAGTGCCCCCCCAGATCTCAAC 859 cg07665217 AGCATGACCAGAATGGTTCCTCAGAATCAGGAGGTGAAACCTAACATTCG 860 cg02505126 AGAGTGGCGCGAGCCTGCGTTTTCCGGCCAGAGGACATGGTGCGTTTTCG 861 cg13565994 GGATCCCAAATAACAGATCTGGGAGAATCCTGCCTCAGTACTTGGATGCG 862 cg17561365 CGCCCACAGGGCCCAGTGGGCTCTGAGTAAGGTGCACGGCACACACCAGC 863 cg09194449 GCCTGGGATCATCCCCAAGTGGGTCTAGGGTGGGCCTGTGAGGAGGAGCG 864 cg26206456 CGACTGCAGGCCTACGGCTTCCTTTGAGGGCTCACGTTTACAAGATAACA 865 cg21462914 CGGCCGCCAACCGACCACTGACTGTCTGCCAGGCATCCGGGCTCCCTCCA 866 cg17560136 GGCCTCCAGAGCTGGGTAAGTGGAGGGCTCTGTGTATGTTGCTGTACACG 867 cg19033875 CGCCCATGCCCGCGCTAGTGGGAGGAGGCTGGAAAGCGCCTTCCCATTTT 868 cg18995788 CGGGCAGATACGAGCAGATTGACTCGCCAGGACTGTCATTGGGCCACCGC 869 cg14387545 CGGCGCTGACGCCGCAAGGCCACTTCTATCGCTCCCGACCACGGTTTGTT 870 cg23083672 CGCTTGTCAGAGTCACCATCTACTACCATGTATTTCAGGCATGGGGCAAA 871 cg02758183 CGAGGTGCTGGTGTTCATACAGATAAGGCTCTCTTCATTGCAACAGCCAC 872 cg20308511 TCATTTGCAAATGTTACTGGGGGACACACCGGCTCCCAGTAGGGTTTCCG 873 cg21412053 CGCAAGAGAAGAGGGAGCACGCCCAGAAGCTGATAATGCTGCAGAACCTG 874 cg26071410 CGCTGTGGCAGCCCAAACGTCAGAGGCACCTCACTTTGCCATGGCCTGGC 875 cg09625066 CGGCCCAGTGAACCTGAGCTTTGGAGTTTGGACGAATAGCTAGTTTACCT 876 cg11235848 CAGGGACTGACGCCCCTCTAGCAAAGCATCTGTGGTACATCCCAGACCCG 877 cg02311013 GATCTCACACTTTGGAGACATACCCCTAAGTAGACCAGAGGCATCACACG 878 cg21664443 CGCCTGGAGAAGCAGTGCCAAGTCTATTCCCTAATAAAGTACTTGTTGTG 879 cg05127178 CGGAGCTGCAGAAAGAAAGGGAAGAAACTAACGTTCTTTCTGCGTCTGGG 880 cg19110434 CGCTAAAACTCACAGCCTGTGGACAAAGCCTTTTTGGTTTTCAAATCGGC 881 cg25697726 CGGTCAAAATTAAAAACTTCTGCACTGAAAAGAAAAAAGGGAGAGAGAGA 882 cg03204600 CGCCCCCAAGAAGCTCCCCACCCCCAAACCAGAACTGGTGGCTGGTGACT 883 cg11049305 ATTGAATGCCAAAGTTACTGGTCCCCAGGCAGCAGCTAGCACATTCAACG 884 cg13501581 CGAGTGGCTACTGTATTGAACAACACAGATCTAGAAACAGAGTGTGGACC 885 cg01564693 CGGGAACCTCCACAGGCTAAGGTGAGTGCCAGCACTGGCACCAGCCCTGG 886 cg10509982 CAAAGGCAGACATCGCTTCAGGTAGGTTGCAGCCAAAATGGCTTCCAACG 887 cg20557017 TTCTTTGTGGCCAAGAAATTCCCACCTTACCACTTACAGGGAGGCCATCG 888 cg05373692 CGCAGTGTGAAAGATGCGAGCCATAGCATCTCGTAACATGAAGGCCATCC 889 cg05470074 TATTTTGTATTTGGCTTGCTCTGGTTTCTGCTGGAGCTTTTAAAAAATCG 890 cg08262933 CGGGATTACCTAGTGCCTTCACAATCGGTCAGAGCTGGATTCAGATTCCT 891 cg15062055 CGGGTTACTACTGTACCTACCAGCTGATTCCAGCAGCCTTTTCTCCAGCG 892 cg12889449 ACCGTTTTTTCCCCCGAATAAAGTGTGCCCGTGTAGCTGTACAGATTTCG 893 cg23955970 TCTTTCTTCACGTCTTGTTGGTTACTGCTACCTGGCTTCCAACTGTTGCG 894 cg05530348 AGCTGGCCAGACTGCTGCAGTTTTCAGTCCCAGGAACTGAGAACGTGGCG 895 cg11595135 GGATTTGGTCTAGGCTTTCTGGCTTGGCCCTGGAACTCTCAGCAAGGACG 896 cg06298190 CGCAGGTTACTTCCTATAAATGGAAGAACCTGAATCATTAAAAGATAAAA 897 cg20686207 TAAGGCCTCGGCCATGACTGTGGGTGGGTTTAACTCAGCTAGCAGCTGCG 898 cg14686949 CGAGACTAAGAAGTGACCCAACCTAGGTGACCCTTCAGCTAGGTCACTCT 899 cg18003791 CGCCCATAAGGATATAAGGGAGACAGAGGGAAAAAGGAGGAAGCTCACTC 900 cg05295197 GCAGGCGGGCCTCAGGGGCTGTCTTTCCTGCACGGCTGTTGTGTGCTTCG 901 cg23106779 CGGCACCACACAGCTAGTGCCGGAACTTTTTGTGCAGAGAAAGAAGACCC 902 cg10143960 GGTAGGGTGGGGCCAGGCTGAGCCTGCTCTGCGGGTCCCGGACCCCAGCG 903 cg07284273 CGTGCAGGCCACAGTAATGGTGGTGCAGGTGGCAGGAGTAGGTGCCCTCG 904 cg17747265 GGACCTGAAGATCTGCATGTATGACCGGCTCTACCAGGACTCTGTGCTCG 905 cg09971184 TGACACTGTCTTCGGGGCACATATGGTCATCTATGAGTGCTGTGAGCACG 906 cg00935435 GAGGGGCTGGCCCCCTTCACCCTGTGATCGACAGGACAGAGCTGGGCACG 907 cg15651727 ATGCATGTTTCACACCTTCTAGAACTCAGAGTAAACCTAGCTCTTCAACG 908 cg13990487 GTTAACAACTGTCAACAGCCTATAGAAGATCAAGGCTAAATTCCCTAACG 909 cg00456299 CGAGTTTAGCTTTTAAAGAATCCTTTGAAGAAGGATTGCAACGTGACTAA 910 cg04974804 TAGAGAAATACATTGTAACAATTTGGTTTCTTTCCTGATGGCCTTTATCG 911 cg13574848 ATTACAAGCATGAGCCAGGGCACCCAGCCAGAAATGATATTTAAACTACG 912 cg25616777 CCATGTATAAGTCCCCAGGACTCACAAATCTCCTCTTTCAGGATATCTCG 913 cg12386614 CGTTAGGTCCCACGGCTTGCCCTGCTCAGCACCTAATAAGGCCATTAGCT 914 cg15355146 CGGGGGCTAAGCCAGCGCCGTCTGCCTCTCCACCTAGACCAGACCCCTTC 915 cg19471466 CGGGGGGTGCCAAAAAGCCCTCCTTCTGACGGCCCTCCTGGAGCCCAACA 916 cg14709691 CGCTTTACGGAAAGAACGCCCCGCGAATAAATCCGGAGCGGATTTGCATC 917 cg21280384 CGGAGCCACAGAGAACACTTTTGAGACCAGCAGACAACCACAGATGTGCG 918 cg14476101 CGCCAGCAGATACAAAGGCAGACAAAGTGAGCGAGGCAGTTTCCAGGACC 919 cg19693031 TTCCTATCACAATTTACAAAAAGCCTCCAAAAAACCTTGAAAAGCTTACG 920 cg16700924 CAAGGGAAGCTCCCGCCTCAGGGTCTCTGTCTTCCTGTGGTTAAGAGACG 921 cg20246707 CGCTCCGGCTGGTAATTTCCAAACACAGAGGGAAGAGCATGGTGGAGCAA 922 cg01365152 CGGAACTGTCCTCGACCCGTGGGTACAGTATTTTCTCGTATGCCACCAAG 923 cg20327105 TTGGTGCAGTTTTGAGCGTCCGTGTATAAGGAGTGGCTCCGTAGTCACCG 924 cg25741192 CCATCAGTAGAGCAGGAGTCGTTGGCTCAATGCAGTGCTGTCTGCAACCG 925 cg13678787 CAGTGTTCGGCAGAGGACAATCTGTTCGAGAATAATTAGATTCCCCCTCG 926 cg08614871 CGGATGCTTAATTACAAAAGGTTTTGCCCCTGTAGTGACCGGGCAGCAAT 927 cg22660341 CAGCTCCCACCTGTGCAGAGTGGCGGCCTGGGAGCTGAGCGTGGCCCTCG 928 cg19647685 CGCGCAGCTTCCTTACAGGAGGCTTTGAATGCTGCCTGGAGTATATTTGA 929 cg03153178 CGCTTAACAATGAGCGGGGCTATATGTCTTGCGCTTGCTCTGAAGGAAGG 930 cg10531748 CGCCGGGAGCTGCTCGCCCAGTGTCAGCAGAGCCTGGCCCAGGCCATGAC 931 cg01508796 CGGGTGAGTGTTTAACTTTCAGCCCAGCTTAAGATCTCCGAAGACACCCA 932 cg06439736 GCAGCGTGCCTACGCTTTGTTGCAGGAGCAGGTTGGAGGGACGCTGGACG 933 cg18853287 AGGATGTTCAGTGACTGCAGCCAGGCCTACCTGGAGAGCTTTTTGGAGCG 934 cg13446658 CGGCTTCTGAACTTTGTACTTTGCGAGTGTACTTTGTACTTTGGTCCTGT 935 cg03322338 CGGCCTCTTATCTCGACAATCGGTTGCTCTTGGCCAGCCCGTCCTGCTGC 936 cg09566331 CGCTTATGCCCAGCTGCTCCAGCTGGAAAAGACAGGACTGCAAGTGGGTT 937 cg21992507 CGGACTTTCTGCGCAGAGAAAACACTATCTCCTTTGGAGGCTGTGCACTT 938 cg01438090 CGCTGATTGCACTGTCAAGAAGGAAAATACTGTTTGAAAAGACAACAGGA 939 cg01562537 AACAAACCATTACGTGTTCCAATTAGAGCGGCAGCGGACCTGGCTGGCCG 940 cg17044529 CGGCAGTGAGTCACTTACATAGATGTGTGTAACATGCCCCAGAGACCTCA 941 cg00574958 AGGCCAGCGAACATCCAGCTGTCAGTTGGTCTGGGGACTATCAGCATTCG 942 cg17058475 AGCGACACTTATGTCCTCAATTTATTGCTACGTTCACTCGGTCAGAGTCG 943 cg21960184 TTCATGCCGGACAAATTTCCCTATGAACAATGCAGCCAAGGGCTGCATCG 944 cg02395454 CGTGGGCAGAGTGGTTGGCTGCCCCACTGTCAGCCTGTTCTGAAAGTAGG 945 cg24675983 TGCAGTTCTTATAATAAACACTTAAACAGGGCCAGGCACTGCAGTTCACG 946 cg21574681 AGGACTAGATGTTCACTTTAAATCCAGCCACTGCTCCAGACTGCAAATCG 947 cg17850642 CGCCCTTTTCCGGTGTCCTGGGCTCAACAGGGATAAAAGGAGGCAGCACT 948 cg01031441 GGTGGGGACTGGCATTAGGGGTGGGCGGGAGAGACCCTAAAAATCCACCG 949 cg01638369 CGTGTGTTTGTAATTGTGTATGGTCCTGCTAATGTTTGTGTTTATCACTG 950 cg07539443 CGGTTCACAGCATGGGAGCATCTGTGCTATACCAAGATTACTCATAGGAT 951 cg15997315 CGGGGCTGTCAAGTGTCTTTAAAAGGTCCCTTCAGGGTGGATGGAAACCA 952 cg13842421 GCCCCCAGCTCTCAAGGCCGGTGCAAGGATGGGCTGTGCTCAGCGATGCG 953 cg02925268 TCCTCAGACTGTTGGGATGATGAGCGGACGGTGAACTCCCACCACCTGCG 954 cg26975476 AGCGGGAGTCACTTCGGAGTGGGGTGTTGACAGCGTGAAGCCCCTGAGCG 955 cg13589330 CGCTTTCAGGGGTCAAAGACCTGGCAGAAATGACTTCCCAACCCCAGATG 956 cg02853801 TAATTGCCCTCCTGTTTCCTAGAAAGCTTATAGGGCAGCAGTAGGAAACG 957 cg06147196 CGCAGAACAGCACAGTGTCACATGGTCTTCACGGCCCTGAGGGCCCAACT 958 cg24126612 CGGGGACCACCAGCTGCTTTGCCCATGGATTGGAACGCATTGGGTGGAGT 959 cg26407029 GCTGTTCCTCATCACTCAGTACCCAGTTTGCTGACGTCTACAATATTCCG 960 cg01802397 CGTGATTGCACGTATCAAGATGTGTGCTCTTAGATTTGTTTTCAATCCAG 961 cg01885071 CGGCGCTCAGCAAATTTAGATGAATGCAACGCTTCAACGTGGGCTCTCGA 962 cg01308343 GTCTTGATGTGACCAGAGAACACCATGTACAGGCAAAGCCCAGCCCCTCG 963 cg15471661 GCTGTCGCTGCAGGTGGGAACCGAGAGCCAGGAAGCCCAGGGAGCTGTCG 964 cg16558846 ACAAGGGGCTCCTCCTTTGTCCTGGGGGAGTTGCATGGATCCTGTCTTCG 965 cg17382302 GTTCCACATAGAGAGCCTGGCTGCATGATCTTGAGGTCACCTAACTCCCG 966 cg25291250 CGCGCCTGGCCTATACAATATTTTAAATAATTTTGTGTGTGATACAATGA 967 cg01021485 CGCCCGCCTCATTTGCGCCTTGCAGCACTGCTGGACCAGGTATCTGCCCG 968 cg27505538 CGGGTCAGACCGTCATTGGCCATCTCACAGTCCCGGCGCTCCAGAGCCCC 969 cg24924577 CAGCCATTGGCTGATTTGAAAGAATACACAGGGTGTGCCAGCTGGTGTCG 970 cg00564555 CGGTCTCAGCCGTCCCCATGGTCTCAGCCGTCCCCAGGGTCTCAGGAGTC 971 cg02193806 CGGTTATATCAACATGATGCCCAAACACCAGGTCTGCTTAGGGTAAAACT 972 cg05732130 GTACAAATGGGTCCAGGAGCTTCAACGAGCGTTCCAGCTTCTTCGTAACG 973 cg02189786 CGCGCACCGCCCCGCTCCACGCCCCTATCAGGATTCGGGTCCTCGTGAGG 974 cg00534468 CCTCGACTGCTGGCTGTGATGTTCTCCAACACTGCTATTCACAGCGAGCG 975 cg03699074 GTCCCGTTCACACTGATCGTTCCATTTCCTCTTGTGTCTGAGTGGGAGCG 976 cg01829657 CGGGGGTTGGTCAGCACCACACACTGTGCACAGGGAAGATGGAGCCTCGC 977 cg00892703 CGCTGATACTGACATTTACAGATGAAACCGTCTGAGACCCGCTTCCCAGC 978 cg27113347 TCGATGCTGGTGGTCTGCGTGGTGGTTCCCAACAGCAGTTCAGGGGCTCG 979 cg09826306 GGCGGCTCTAGCCTCTCGGGGGACGTTCGGCTACTCCAGTGGGGGGTTCG 980 cg25567048 GAACAGCCACGCATGGTCCGTGAAATGGGATCATGTGCAGAACAGCCACG 981 cg06513864 CGCCCCACCAGCCCGAGAGCTAGAATTGAATCTGGGGGACCCCTGCGGCG 982 cg11020638 AGCACTGTAGGGAGTGGCCGGGATCATTCGAGCCCACAGATAGTGCTCCG 983 cg05815247 CGGACAGCTAATCGTTTTAGTGACAGGATGAGAGAGCCCTTCGTGTTCTG 984 cg21348173 CGGCCTGACACTCTCAGAGATGGGATTCCCAATCCATCCACTCTGAAATG 985 cg22850234 CCGGAGGCTGATGCCTTATCCATTAGGCCACTGGGTCACCACAGGATCCG 986 cg10508317 AGCTGAAGGTCTTGAGGCGCAGGCTGGTGTCCAGGGGGCGGCTCATCCCG 987 cg27637521 GGAAACTTGCTGTGGGTGACCATGGCGCACGGAGCCAGCGTGGATCTGCG 988 cg16252995 CGGCAGAGAGGACAGGATCCTGCCACCCAGCCTCATCCCCATGCTTCCCC 989 cg03524223 TCCCACCTTGTGAAACTTTGCCAGGTGTCGCTCACAGGGGACTGGGTCCG 990 cg05495454 AGAACATCCACTCTCTTCCAGCCAGAATACCATTAATACGTGTGTCAGCG 991 cg07573872 CGGGAATGGTGACTCAGCCTTCCAGGAACCTGCGTGGCGTCTGTTTTTTT 992 cg22519313 CTTCCTATGTGATTTTCTTCCAACATTCTCCCATGAAAAAATGTCAGACG 993 cg05313771 TGGGTGATGGAAACTGAGGCACAGTCATCCCCCCAGCAGGTCCTTGGACG 994 cg09522532 CGGGCCCCCCAGAATGGAACACGAGGGGGTGGTCTCTTGCTGGGCTCCAG 995 cg15783941 CGCCAGCCCAGGGCTGGATGTTTTATTTTGGCATGCCCCAGGTGCTCTTC 996 cg24115922 CGCACATGCGCATCCAACCCGATTCCACGGGTTCCCAGGAGGCGGATTCC 997 cg18593194 CGGGAAGTCTGCCAGTTGGCAGTCTCTGACGTAAAAGATAGCAGATACTG 998 cg25654926 AGCCCCACTAATAGATATTCTGATTCTGTTGGTCTGGAATGGGAACCGCG 999 cg10853231 GGAGGGCAGGTCCGGGGGCTCTGTGTCCCCACTGGGCCCCCAGCACTGCG 1000 cg19717326 CATTAAAGCTCAGACTCGGGTTTTCCGAGCAGGTGTTGCAAGCCTAGCCG 1001 cg13650654 CGGGTCTCTAAGATGTCTTATGAATGCAGGTCAGAGGGTCACATGTTAAC 1002 cg02875404 CAGTGGATAGTGACCAACTCTGTGTTGGTCGCCTCATGTTTCCCAGCACG 1003 cg14889658 AGCATTCGGGCATCAGAGGTTTGTGGAGAGGGCAGCCCAGTATACAACCG 1004 cg02591356 CGTCTAAATTACTTGGAGTTGATCTTTCTAAACAGGATTGCTCAGACACA 1005 cg26471058 GCCTGAGGTCAGGAGTAAGCAGACACCAGCACTGCTCTTTCTCCAAGACG 1006 cg25939861 CGGAAATCAGCTTGGGGGCCTTCTAGCCCTGCAGCTCAGAAAAGTGTCAG 1007 cg03913456 ACCGCGCTCAGCCCGTGGAATTTACTTTATAATGGGGAGGCAGATGATCG 1008 cg24506130 CGGTTGTCAGCTGCCGGAAGAAGAGCGTGGAATTGAGAACACAGACCCCT 1009 cg26009035 TGGGTGCCAGGGGGATTATCAGCGTCTGCGCAAGGCCGATGGTGGCAGCG 1010 cg05308744 TGATTGGATGCAGGATTGGCGGGAAGGCAGCGGGGTCATCAAAGGCTGCG 1011 cg22663545 CGATGAGCTCAGCCCCCTCAGTCCAGGCCGAGAGCCAGATGGGGGTCACG 1012 cg02767242 GTCCCCCTCGTTTCCTTTTAGAAGTCATTGGCCGATCGTTTTAAAAGTCG 1013 cg13603203 TGGGATCAGGATCATGGTGAGATGTATGCCTTTCCCAGGAATGCTAGCCG 1014 cg20951650 ACTACTCTCCTGGAGTGGTTACAGTTAACCAAGGCAAAACCCCTTCTTCG 1015 cg12683410 CGGCTGTTGTCCTGCTTACAGGACTCCAGAGCAGCAGGAAGGAGCTATGT 1016 cg16887334 CGCACTCGGCCTGACCCACGGCGACCCTCTGTGACCAATCATACTACCAA 1017 cg26126740 TATTCCAATTTGGGCCTGCGTGTTCGGAGCTCCTGGTGGCGGTCTCCACG 1018 cg10120040 CGGGCTTTGGTGACCAGGTCTTCTGTCAGTGAGCACACATAGCCCGGAGG 1019 cg25761955 CGCCTGTGTGGACGCTTGTGAGCTGCACCTTCATGCTATTATAATGGCGA 1020 cg22871721 GAAAACACACAGATGTCAAACCAGAGGAGCTGCCAAGCTGGGTACCGACG 1021 cg22778797 CGCCGGCCCCAGGTAGTATATTTTTATTGGCACAACTGCCGGTATTTGCC 1022 cg27243685 CGGAGGATGTGTTTCCAGAGAGCTGGTCTATTTCAGACTGACAGGCCACT 1023 cg06500161 CGGCGGGACTAGTTCCTTTGGGCATAATTTAGGTGTTTTGTGAAATGTTA 1024 cg17215151 TGCTTAGTAGCTAAAGGCCACCTTCTATAACACGAAATAGTCTACAAGCG 1025 cg01127300 ATGCCTTCTTGGGGCCTGGCTTGCTGCTGACGGGAGCACTCAGGAATGCG 1026 cg12511214 CGACAACAGGGACTCGGTGGACCTGGCCGAGCTGGTGCCCGCGGTGGGCG 1027 cg07031797 ACAGTCGAAGCGCACTGCTCTGGTGGGGGATGTTGGCGGTGCAGGAGGCG 1028 cg18405719 CGAGGCTAAAATGTGGGGCCAGAAGATAAATCTAATGAGAGCTGTTGCTA 1029 cg21614211 CGGGGGTGTGTTTCTGTTTGTGTAACTGGGTGTGTGTGTATCTAGCCATC 1030 cg06813250 TAGAAACTGATGTTATTCATAGATTCCACACATTGTATAGAAGAACATCG 1031 cg15705999 CACCCCCAGCCTCGCCACGGCTCGCTCCCCCCGGGGTCAGGCCGTTACCG 1032 cg09000779 CGGGACTGTGGCCAAGGCCCTCAGAGGCCATGGGGAGACACGACAAGACC 1033 cg12473849 CGCAAGCACATAACAAAAGCCAAGTTAAGAAGGTTAGCGATTTAGTTGTC 1034 cg27336439 TCACCATTGCCACTTAATGGTCTGAGGGGGGCTTTTCCTGTTACTGCACG 1035 cg00480331 TGGATCGCCCAGGGGCGGGCAGTCCCTGGAAGACAGGTACGGAGTGGTCG 1036 cg13724379 GGAAACGAATCTGGGAACGAGTTTGAAGCGCGGTCAAGTTTGACTCATCG 1037 cg15012625 CGAGGGTCCCAGTGTTCCACATCCTTGCCAACGCCTGTTATTGTCTGTTG 1038 cg01658265 CGGGGTAACCCCAGGTCCCAGTGATTTCCATAGACAAAAGAGATGCTGGT 1039 cg00252095 AGTCTTGCCTGGTTACAGGCCTGTGCTCTCCAGGCAGAACAAGCGCAGCG 1040 cg26805150 GACAGTTCTTGGGAAGCCGGCAAACTGCAAAAGCCCCATATTTCCTTCCG 1041 cg02373784 CGGGCTCCTCAGGTCTGAGGCAGCTCTACCACTGACTTGGTGAATTTTAG 1042 cg07951602 CGCCTCTCAAGAGCACGATGTAAGGGCTCCAAGATGAGTTTGGGCTTCCC 1043 cg19719391 GGCACTTTTCAGCTCTAATGCTCTTGCTTTCCAAAAGCAAAACAGAACCG 1044 cg08284371 CGGGGCAGCACAGACACCCGGGCAAATTCATGATCTCTACCGAGTAAATA 1045 cg26816491 CGTGTCCTTTAAGAACTGTAACACTCACCGCCAGGGTCCACGGCTTCATT 1046 cg06690548 CGTGAACTACCCAGATGCAGCATCCGCTGTTGGTGTGATCTGGAACACCC 1047 cg18376497 CGGAAGGCTGAAGGGGGAGATAATGAGCTAAAGAACAACTTTCACCACCC 1048 cg06176987 TTTAGTTTCCTTCTTTTATTTTCCTGGCAATAGCCCACCATTGTATATCG 1049 cg22744079 CGGACACTGAGGCCTTGTAATGCTGTTGGCTGAAGAGAGGTGGTTGGTCA 1050 cg16400903 TCCGCGGGGCCTGAGCAGAGTCGGCCTTGTAAGGCAGAGGTCTCGGTGCG 1051 cg09884076 CGGCCTCTTGTCCACCTTGCCCCGAAGGCAAGTCTGGATATGAAAAGAAA 1052 cg12599971 AGCTTCTTTGCTTTGACTGTTTTCCTCTTGCTGCGTCTCTTTGGTCTTCG 1053 cg26403843 CGGATTGCTGCTTCTAAGCCTGAGAAGGTCATTTAATGAACTCATTGAGG 1054 cg07017437 CGCGCCAGCCCATGTATGCCATGGAAATATGGCTGTTGGAATTCTGGTGT 1055 cg21036336 CGTCTTGGGGACGCTATGGCTCCAGTTCTGACACTCAAGAAACGATGGAT 1056 cg03714531 GGGCGGCTTCTGATCTCTTGCGGACAGCTCAGATTAGTGGCTCGAGGCCG 1057 cg23036136 CTTGGGTGCCCAGGTCTCTTCCTTCCTGAAGAATGCGGCACTTGCTTCCG 1058 cg10189661 CGGGCAGACTGCGATGAAACCCCAGTTTGTTGGGATCTTGCTCAGCTCCC 1059 cg05218245 GGTTTCCAGGTAGCTTGATCTGTTCCAGGCCTGGCTGGGGTCTGCCCTCG 1060 cg04018738 ACCCACCTGCCCGCCGGATCTGGAATAATGTGACTCGCTGGTTTGTCACG 1061 cg02115397 TGCTGCGGTTGGAGCGCTCCACCAGGATGTCCAGATGGGCTTTGATTGCG 1062 cg12927358 GGGGTTTAAGGATGATTCCCCACTCCACAGCCTGACGCATCGACTCTGCG 1063 cg05466385 CTACGAGTCTTATTTCCTGTTAACTTTTTTCCTTTTGCTTGATCCTATCG 1064 cg01702009 CGGCTGGGGGAGGAGGCACCCACAAGACTGCCTTCTAGGCCACAGGCCCA 1065 cg06560379 CGGAGGGAGGGAGGAACTGCTCTGGAAATCCCCTAGGCCAGGGCCTGCTC 1066 cg21710255 TGTTCCTGTTACTCTTCGTGAATTTTCTCACCATAAAACTGTTGCCAACG 1067 cg20459712 GTACTTTGATGCTCTACTTGGCAGAATGTATTGCTTCTGGTACCCACACG 1068 cg18718984 GTGCTACCTAGAGAGGCCAGCATGTGGGCTCCACATTTCTGCTGAAGACG 1069 cg03707967 CGGTGCTTATAATACATTCCTGTGTGTGTGTTCACTTTGATTTACGGAAA 1070 cg02027561 CGTCAGGTTTATATCTTTGGGATTTGAATGTAAAGATGTTGGATTTTTTT 1071 cg00695799 CGCTCCATGTGACAGAAACATGAGCGTTTCCAAAATGAAAACACTGGGGG 1072 cg18116267 TTCCCTTGCTTTTCCCAGCTTCCGGTAGCCCAGGCATTCCTTGTCTTGCG 1073 cg17501210 CGCCCGATTCAGACAGCTGGACTCAGAGGGATTCTGCTCCACAGAGAAAC 1074 cg26905845 CGGCTAGAGGATGACCCATCTTACGCCTATGGAGAACACTGCGGAAATCA 1075 cg03730314 TTGCCTGTGTGTGATTTATGCATGTTCCCACCAGCAGGTGTGGATTCTCG 1076 cg03394401 CGGGCTCCGATCTCCGGTGTCACAGCAAAGATCCGCGTCTTTCCGATGAA 1077 cg19196326 CGCCTCCCCACGCACTTCCCTGTATGGGTTACAAAGCAGGTGCACATCCA 1078 cg13550401 CGTGGCACACAGAGCCAGGTTTCCCCGAAGTAGTCTAACGCTGTTGCTAA 1079 cg08549335 CATTGGCTCTTCACTTGTTGCTTGGTGTTTTCCTTAGTCTTCTGAGATCG 1080 cg17220237 CGGGACATGAAGTCTAAGGGGATTATTTTGGAGCTTTAAAACGTGATGAC 1081 cg22322818 CGGGAATTTGGTGAGAGTCCCCCACAGTTCTGTCTGCCAGGCACTGGCGA 1082 cg08695830 CGCATCCTCCAGCCATGCTATGCTTGCAAAGATGTCTCTGGTCATTGGGA 1083 cg17981101 CGCCCCAAGTCATGAGGGAAGCTTCCAGACTGACTCAGGCTCAGGCTGGC 1084 cg01337813 GGAAGTCCTCTGTGCATGCGTGTCTGTGCCATAGGCGTGGGAACACCTCG 1085 cg25977769 CGCCTAGGTATGGGAAATGTAAGTATCCTCAGCAGAGACCACAGTTCTCT 1086 cg07592681 CGGTCAGCACTGGGTGAATGGGTTGAGGGAACCCACATAAAATCCCCAAG 1087 cg09584650 CGATCTTCCCCATGGAAGACATGCACATGGGTTAGGAGAATACAACACTC 1088 cg01070197 CGCAACAACTTCTCAGCTACAGTAAATCAGGATTTGGGGTGATAGGAAGG 1089 cg17744279 CAATGTATACTATAGCAAGTGGTACCAGTGCTGCGGGTACCAGTGCTGCG 1090 cg13518625 CACAGATCAGACTGCTAAGTCTTTACACATCCGAAAGTTAGACTTTGACG 1091 cg03393241 GCCACGGGAGATTTCATCAGAAGAATATCAGTCTATTGATATGGGTGCCG 1092 cg22660904 CCTCCCTACAACTGTAGGTTCCCCTGCTGCTGGGCTCAGCTGGGAGCTCG 1093 cg26074100 CGGGACAGCCCCGATGCCGATGCCTGTGTTCTGTGTCCCCAGGTCTAAAG 1094 cg01029867 GAGCCCAGCGGGGTTAGGGTTCAGGTGAGGGTTTAGGGTCGGGGCGAGCG 1095 cg14268714 CGTACCCCGGGATCTGGCATTTAGCTGGCAATCAAATATGTGGTGAAACG 1096 cg13673536 GTTTAAAATTCTCTCTTCTGAGTAATAGTGTTCATGTGCTCCCATTCTCG 1097 cg14457452 ACTAGGTAAACACCACAGTAATAACTGTTGCAGGTAAGAATCATCAGGCG 1098 cg21208062 CGGGAAATGACCAAGCGTCTCCACAATTTAGGGGCAGGTCAGGGTACCTT 1099 cg12020464 CGCCACAAACCCACCAAGGTGCTCTCCCCCGAGTACTTGTGGGACCAGCA 1100 cg13873209 CGGCACCACTCATGTTCATAGGCCAGCTGAGGCTCAGGGGGCTGACTTCC 1101 cg13499318 CGCTGGGCAACCGTAAGTATTTAGGGAGACAGAGGTGCCTCTTCTGCAGA 1102 cg13915886 CGACGCCCTTGCTCACAGGCTCCATTGAGAGGCTGCCCCGGTGCTCATCT 1103 cg22249566 CGCAGCCCTGCGGGACCTGAAAAGGCGAGACTAAGTCACCAGCCAAGCTG 1104 cg17511128 CGGTCACTGTTTGTCTTGAAAAAAAGAGAGAAAATGTTTGTCATTCCAGG 1105 cg02495445 CGTCTTCTAGCAGGTATGAGGGAGACAACTACAAACGCCAGAGGCTCAGC 1106 cg16088894 AGCTCTCGCCGCTGAAGGGGCTCAGCCTGGTCGACAAGGTGAACAGGCCG 1107 cg13573587 GCAGGGCAGCCAGCTACGAATTTTGAGCTCTGGGGCGCGCTCACCTGGCG 1108 cg03788610 CGGGTCAAGAGCAGTGACCTAGTGGCCCTCAGCCCTGGATGGAGGACAGA 1109 cg03990195 CGCCATTCTGTTGGTACTAAGGTGTTGTGGTTGGTAGAAGCTTAAGTGAG 1110 cg08147391 CGGGTCGTGGTGACTACTAATGGGTATAAGATTTCTTTTTGAGATGATGG 1111 cg05548952 TGTTGACCTCAGGTCCAGTTTGTTTGCAGCCTCCCTCTTCTGACAGATCG 1112 cg07929447 TTGTCTCAGATTACAGGAACCAAATGCATGTCTGGCCTTGTGCTGCTTCG 1113

EXAMPLE 2 REFERENCES

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All publications mentioned herein are incorporated herein by reference to disclose and describe the methods and/or materials in connection with which the publications are cited (e.g. Lu et al., Aging (Albany N.Y.). 2019 Jan. 21; 11(2):303-327. doi: 10.18632/aging 101684). Publications cited herein are cited for their disclosure prior to the filing date of the present application. Nothing here is to be construed as an admission that the inventors are not entitled to antedate the publications by virtue of an earlier priority date or prior date of invention. Further, the actual publication dates may be different from those shown and require independent verification.

CONCLUSION

This concludes the description of the preferred embodiment of the present invention. The foregoing description of one or more embodiments of the invention has been presented for the purposes of illustration and description. It is not intended to be exhaustive or to limit the invention to the precise form disclosed. Many modifications and variations are possible in light of the above teaching. 

1. A method of obtaining information on one or more physiological factors associated with an age of an individual, the method comprising: obtaining genomic DNA from the individual; observing methylation of the genomic DNA in at least about 42 methylation markers from the group of methylation markers in SEQ ID NO: 1-SEQ ID NO: 1113; correlating observed methylation in the methylation markers with the one or more physiological factors associated with the age of an individual such that information on the one or more physiological factors associated with the age of an individual is obtained.
 2. The method of claim 1, wherein the one or more physiological factors associated with an age of an individual comprises a number of years in which the individual has smoked in their lifetime, the method comprising: observing methylation of the genomic DNA in about 172 methylation markers from the group of methylation markers in SEQ ID NO: 1-SEQ ID NO: 172; correlating observed methylation in the about 172 methylation markers with the number of years in which an individual has smoked in their lifetime, such that information on the number of years in which the individual has smoked in their lifetime is obtained.
 3. The method of claim 1, wherein the one or more physiological factors associated with an age of an individual comprises plasma protein levels of tissue inhibitor metalloproteinase 1 in the individual, the method comprising: observing methylation of the genomic DNA in about 42 methylation markers from the group of methylation markers in SEQ ID NO: 173-SEQ ID NO: 214; correlating observed methylation in the about 42 methylation markers with plasma protein levels of tissue inhibitor metalloproteinase 1 in an individual, such that information on plasma protein levels of tissue inhibitor metalloproteinase 1 in an individual is obtained.
 4. The method of claim 1, wherein the one or more physiological factors associated with an age of an individual comprises plasma protein levels of Cystatin-C in the individual, the method comprising: observing methylation of the genomic DNA in about 87 methylation markers from the group of methylation markers in SEQ ID NO: 215-SEQ ID NO: 301; correlating observed methylation in the about 87 methylation markers with plasma protein levels of Cystatin-C in an individual, such that information on plasma protein levels of Cystatin-C in an individual is obtained.
 5. The method of claim 1, wherein the one or more physiological factors associated with an age of an individual comprises plasma protein levels of Beta-2-microglobulin in the individual, the method comprising: observing methylation of the genomic DNA in about 91 methylation markers from the group of methylation markers in SEQ ID NO: 302-SEQ ID NO: 392; correlating observed methylation in the about 91 methylation markers with plasma protein levels of Beta-2-microglobulin in an individual, such that information on plasma protein levels of Beta-2-microglobulin in an individual is obtained.
 6. The method of claim 1, wherein the one or more physiological factors associated with an age of an individual comprises plasma protein levels of growth differentiation factor 15 in the individual, the method comprising: observing methylation of the genomic DNA in about 137 methylation markers from the group of methylation markers in SEQ ID NO: 393-SEQ ID NO: 529; correlating observed methylation in the about 137 methylation markers with plasma protein levels of growth differentiation factor 15 in an individual, such that information on plasma protein levels of growth differentiation factor 15 in an individual is obtained.
 7. The method of claim 1, wherein the one or more physiological factors associated with an age of an individual comprises plasma protein levels of adrenomedullin in the individual, the method comprising: observing methylation of the genomic DNA in about 186 methylation markers from the group of methylation markers in SEQ ID NO: 530-SEQ ID NO: 715; correlating observed methylation in the about 186 methylation markers with plasma protein levels of adrenomedullin in an individual, such that information on plasma protein levels of adrenomedullin in an individual is obtained.
 8. The method of claim 1, wherein the one or more physiological factors associated with an age of an individual comprises plasma protein levels of Leptin in the individual, the method comprising: observing methylation of the genomic DNA in about 187 methylation markers from the group of methylation markers in SEQ ID NO: 716-SEQ ID NO: 902; correlating observed methylation in the about 187 methylation markers with Leptin in an individual, such that information on plasma protein levels of Leptin in an individual is obtained.
 9. The method of claim 1, wherein the one or more physiological factors associated with an age of an individual comprises plasma protein levels of plasminogen activator inhibitor 1 in the individual, the method comprising: observing methylation of the genomic DNA in about 211 methylation markers from the group of methylation markers in SEQ ID NO: 903-SEQ ID NO: 1113; correlating observed methylation in the about 211 methylation markers with plasminogen activator inhibitor 1 in an individual, such that information on plasma protein levels of plasminogen activator inhibitor 1 in an individual is obtained.
 10. The method of claim 1, wherein the one or more physiological factors associated with an age of an individual comprises predicted age or lifespan of an individual, the method comprising: observing methylation of the genomic DNA in about 1113 methylation markers from the group of methylation markers in SEQ ID NO: 1-SEQ ID NO: 1113; correlating observed methylation in the about 1113 methylation markers with predicted age or lifespan of an individual, such that information on predicted age or lifespan of an individual an individual is obtained.
 11. The method of claim 1, wherein genomic DNA is obtained from human fibroblasts, keratinocytes, buccal cells, endothelial cells, lymphoblastoid cells, and/or cells obtained from blood, skin, dermis, epidermis or saliva.
 12. The method of claim 1, wherein: methylation is observed by a process comprising treatment of genomic DNA from the population of cells from the individual with bisulfite to transform unmethylated cytosines of CpG dinucleotides in the genomic DNA to uracil; and/or genomic DNA is hybridized to a complimentary sequence disposed on a microarray.
 13. The method of claim 1, wherein correlating observed methylation in the methylation markers comprises a regression analysis.
 14. The method of claim 1, wherein genomic DNA is amplified by a polymerase chain reaction process.
 15. A method of observing the effects of a test agent on genomic methylation associated epigenetic aging of human cells, the method comprising: (a) combining the test agent with human cells; (b) observing methylation status in at least 42 of the methylation markers of SEQ ID NO: 1-SEQ ID NO: 1113 in genomic DNA from the human cells; (c) comparing the observations from (b) with observations of the methylation status in at least 42 of methylation markers of SEQ ID NO: 1-SEQ ID NO: 1113 in genomic DNA from control human cells not exposed to the test agent such that effects of the test agent on genomic methylation associated epigenetic aging in the human cells is observed.
 16. The method of claim 1, wherein the method comprises: observing methylation of the genomic DNA in about 172 methylation markers from the group of methylation markers in SEQ ID NO: 1-SEQ ID NO: 172; observing methylation of the genomic DNA in about 42 methylation markers from the group of methylation markers in SEQ ID NO: 173-SEQ ID NO: 214; observing methylation of the genomic DNA in about 87 methylation markers from the group of methylation markers in SEQ ID NO: 215-SEQ ID NO: 301; observing methylation of the genomic DNA in about 91 methylation markers from the group of methylation markers in SEQ ID NO: 302-SEQ ID NO: 392; observing methylation of the genomic DNA in about 137 methylation markers from the group of methylation markers in SEQ ID NO: 393-SEQ ID NO: 529; observing methylation of the genomic DNA in about 186 methylation markers from the group of methylation markers in SEQ ID NO: 530-SEQ ID NO: 715; observing methylation of the genomic DNA in about 187 methylation markers from the group of methylation markers in SEQ ID NO: 716-SEQ ID NO: 902; observing methylation of the genomic DNA in about 211 methylation markers from the group of methylation markers in SEQ ID NO: 903-SEQ ID NO: 1113; and/or observing methylation of the genomic DNA in about 1113 methylation markers of SEQ ID NO: 1-SEQ ID NO:
 1113. 17. The method of claim 15, wherein a plurality of test agents are combined with the human cells.
 18. The method of claim 15, wherein the cells are primary keratinocytes from multiple donors.
 19. The method of claim 15, wherein the method observes human cells in vitro.
 20. The method of claim 15, wherein the test agent is a compound having a molecular weight less than 3,000, 2,000, 1,000 or 500 g/mol. 